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BIO 310
Midterm 2 Study Guide + Final Exam Questions
SBU
Summer 2013
N/A

Su13 BIO 310 MT2 review:

Covers lectures 7/18, 7/23, 7/25, and 7/30: ch 15-20
1.Define gene expression
Information in the Genome and how it is acted upon. Genome is the cookbook and
Protein is the meal. Gene Expression Shopping and Cooking, Information on
how to make proteins, But proteins have to be made. Involves a number of
Steps, making RNA copy then Translating that mRNA into protein. IN
Eukaryotic Cells you have to make primary Transcript that has to go through
some events that are RNA processing so it is in the mature RNA FORM and
ready to be exported out of the nucleus and into the cytoplasm and Recognize
by the Ribosome. All of these are part of Gene Expression !!!!.
2.Distinguish how the three levels of regulatory DNA elementspromoters,
promoter proximal elements, and enhancerscontribute to the
regulation of expression levels. These Regulatory DNA elements have
proteins that recognize them, they help regulate the assembly of a
Transcription pre-Initiation Complex at a certain promoter. These DNA
and proteins work together to turn on Gene Expression.
The Transcription Unit contains DNA Sequences elements that tell RNA
polymerase where to start and finish transcription.
Promoter: Assembly of DNA Sequences required to form a preinitiation complex
and initiate transcription. Promoters promote Transcription, they help RNA
POLYMERASE sit down and face the rite way so RNA polymerase knows which
strand to read. RNA POLYMERASE 2 can only transcribe the strand that is in the
3 to 5 direction, its making RNA POLYMERASE in the 5to 3 direction. And
double stranded Nucleic Acids are in the antiparallel direction.
Promoter Proximal Elements: Are Short (10bp), and within a few hundred base
pairs from the promoter (IN PROXIMITY TOO) Close to initiation of transcription
Examples of Promoter Proximal Elements: SP1 is an example of an element found
in constitutively expressed housekeeping genes their products are involve in
normal cellular functions. GRE is a regulated element by (glucocorticosteroid).
Enhancers( Distal Elements): Can be much further away (up to 10kbases) and
they can be downstream as well as Upstream from the promoter, and they can work
in either orientation relative to the promoter.

Enhancers work in Similar WAYS to proximal Elements in that they are specific
sequences that bind to specific proteins. Enhancer Elements provide a further level
of gene expression. So Enhancers are farther away, are on either side of the
promoter work in either orientation relative to the promoter. Most enhancers
work in a tissue specific fashion, by interacting with Tissue-Specific Activator .
proteins. Enhancers promote the formation of Complexes contacting DNA binding
domains. Activator proteins bound to an enhancer attracts RNA polymerase and
GTFS to the promoter. Looping of DNA permits contact between activator and
transcription complex at promoter. Mediators: Proteins that assemble at the site
of the Enhancer-Activator somehow bend DNA in such a way. Proximal Elements
and Enhancers are control elements, which are segments of Non-coding DNA that
help regulate transcription by binding certain proteins. Control Elements and the
proteins they bind are critical to the precise regulation of gene expression in
different cell types.

3.Explain how DNA elements and transcription factors are used in a

combinatorial strategy to regulate the initiation of transcription.
So first Chromatin-Modifying enzymes provide initial control of gene expression
by making a region of DNA either more or less able to bind the Transcription
machinery. To initiate transcription, eukaryotic RNA polymerase requires the
assistance of proteins called Transcription factors. Transcription factors bind
specifically to specific DNA sequences there is a specific interaction between
R-groups of amino acids and the reactive portions of the bases with a specific
DNA-protein interaction. So transcription factor recognizes promoter or
Regulatory region of their target genes and ignore the rest of the genome. So
in response to whatever signal activates Transcription factors it turns on a
subset of genes , and all of these genes have the conserved recognition sequence
that the Transcription factor recognizes.Transcription Factors have two domains
one of them is a DNA binding domain that recognizes a particular sequence of
DNA and another domain is going to be a trans activating domain the trans
activating domain recruits the whole Transcription initiation complex to come
to that site . So specific Transcription factors recognize their specific sequence
and then try to summon the whole Transcription Complex to come to them.
Transcription factors are modular proteins, they have a DNA binding
domain that recognizes their sequence and a trans -activating domain that helps
recruit Transcription Complex. Amino Acid side chains Hydrogen Bond with
groups on the edges of bases in the major groove. These interactions are
stabilized by contacts with deoxyribose and phosphates.

4.Know the basic classes of transcription factors (Zinc finger, leucine

zipper, homeo)
Transcription factors have DNA binding domains which typically have an alphahelical region that can interact with the major groove, interacting with the
exposed groups of that particular sequence.
Homeodomain: Is a 60 amino acid motif that was discovered in Drosophila
proteins that regulate development. They are found in many eukaryotic
transcription factors, including 150 in humans. Helix-turn-Helix in major
groove that recognizes 6 Base pairs. Additional binding affinity is provided in
the Home domain by a flexible arm that interacts with the minor groove. It is
able to nescle itself into the DNA Sequence it recognizes by having two alpha
helical regions at an angle to one another that is similar to the angle of the DNA
double helix. Only portions that can nescle itself into the double helix while its
turning is going to have a ROBUST impact on the structure of DNA in that
region and facilitate Transcription. There are going to be particular R groups
that are driving interaction with the particular bases and Reactive portions of
the Deoxyribose. The Homeodomain is folded into 3 alpha helices, which pack
tightly to one another by hydrophobic interactions. Asparagine contacts an
Adenine.
Zinc Finger: Are found in more than 600 Human Transcription factors. The
fingers are 30 amino acid with conserved Cysteine and Histidine Residues that
bind Zinc ions. They recognize 3 conserved 3 bp sequences. Zinc fingers have
the same basic structure but recognize different DNA sequences, because their
amino acids differ. The Zinc ion helps keep the the transcription factors 3d
shape. Zinc finger is going to fold itself in a particular way that is achieved by
putting the Zinc ion in there that is attracted to Cysteine and Histidine exposing
a particular portion of the protein to a particular sequence of DNA.
Leucine Zipper: Leucine rich regions are Hydrophobic, Leucine has just
Hydrocarbons in its R group. If you have two Leucine Rich Regions they are
going to stick to each other. Because they are Hydrophobic they are not happy
in aqueous environment of the Nucleus. So they zip themselves up , they
interact with one another in a way that excludes water and it is a stable
interaction. Leucine Zipper brings together two molecules and then allows the
rest of the protein to interact specifically with DNA. The Leucine Zipper consist
of two motifs: a basic region that recognizes DNA(short inverted repeats), and
leucine rich regions that form coiled-coils, stablilized through
hydrophobic(leucine) domains . The Leucine Zipper 2 alpha helical DNA
binding domains. Each binds to one half of a symmetric DNA structure. The
3

two helices can dimerize by forming a coiled coil, which is stabilized by

hydrophobic interactions( Leucines pointed towards one another hold the
helices together zipper Side chains from both helices strand into groove to
contact DNA BASES. This specific Interaction between side chains and bases
are Hydrogen Bonds. One of the features of the Leucine Zipper is that it can
Heterodimerize Heterodimer is a Combination of different sequence elements,
its just another way of adding to complexity of gene expression. The
Transcription factors can heterodimerize, which can alter their DNA specificity.

5.Know how transcription factor activity can be regulated.

Regulation of transcription factor activity: Some transcription factors are
synthesized as needed. (De-Novo Synthesis) Some exist in the cell all the time
but are inactive; they need to have a ligand bind to them for example Hormone
Response (Estrogen Receptor). Other transcription factors exist in the cell all
the time but need to be activated by phosphorylation. Perfect example would be
p53 gene which is responsible for cell cycle arrest and DNA repair (Requires
post-translational modification to become active) Other transcription factors
need a partner to be active. Other time transcription factors are autoinhinbited
and they need to be dissociated from inhibitor. Some Transcription factors are
sequestered in the cytoplasm, Transcription factor is not useful in the
cytoplasm, but that is how its regulated, under certain conditions it go inside the
Nucleus and turns on gene expression
List of ways of Regulating Transcription factor activity:
1. DeNovo Synthesis(synthesized as needed)
2. Ligand Binding (inactive in absence of ligand)
3. Phosphorylation (Requires post-translational modification to become active
ex p53 genes responsible for cell cycle arrest and DNA repair.)
4. Heterodimer Formation( Needs a partner to be active)
5. Dimer dissociation( Needs to be dissociated from inhibitor)
6. Subcellular localization (Trapped in the Cytoplasm until needed in the
Nucleus)
6.Understand the role of histone modification and chromatin accessibility in
transcription.
Chromatin modifying enzymes provide initial control of gene expression by
making a region of DNA either more or less able to bind the transcription
machinery. Histone modifying enzymes can interact with Transcription factors

to help sustain transcription or eliminate it. There are Nucleosome Modifying

Complexes or Chromatin Modifying complexes that acetylate or DE acetylate
or Methylate DNA and affect the way Nucleosomes are folded and help slide
Nucleosomes along chromatin. Transcription Activators can recruit
HAT(Histone Acetyle Transferase) . Transcription Repressors can recruit
HDAC. Modifications of the N-term tails regulate the ability of Nucleosomes to
block or promote access of the transcription machinery to the DNA.
Nucleosome remodeling complexes use ATP to alter nucleosome location, by
destabilizing interaction between DNA and Histones. Histone Modifying
enzymes are part of Complexes that are recruited to chromatin by interaction
with regulatory proteins. Bromodomains interact with acetylated tails,
chromodomains interact with methylated tails. Enhanceosome for example
multiple factors binding lead to bend in DNA greater stimulatory effect of
binding. Removal of the Acetyl groups from H3 and H4 histones is among the
initial steps in conversion of euchromatin into Heterochromatin. Histone
deacetylation is accompanied by methylation of H3K9 histone
methyltransferase. Once HP1 is bound to the Histone tails,HP1-HP1
interactions facilitate chromatin packaging into Heterochromatin state.
Transcription Regulators can initiate Transcription and can also make
environment more favorable for sustained Transcription by acetylated Histone
and driving Chromatin into open conformation. If you recruit Chromatin
Remodeling enzymes that change the conformation of Chromatin by acetylating
the Histone tails and opening it UP then it can make an impact on long-term
regulation of gene expression.
7.Explain the techniques used to identify DNA sequences that bind
regulatory proteins, and to identify the regulatory proteins themselves.
How scientist identify Regulatory Regions in Upstream portion of gene where
Transcription factors can bind.
Methods for Identifying, isolating and localizing transcription factors include:
DNA foot printing
Phylogenetic foot printing
Gel Shifts
ChIP Chromatin immunoprecipitation
DNA foot-printing: The footprint is the portion of DNA that is protected from
Nuclease activity by a specific interaction with a protein. The nuclease can be
specific (restriction enzyme or non specific) So when a protein binds to DNA
5

tightly it is sort of like making a footprint and you can see places where protein
binds to DNA. So if you do a Gel Electrophoresis the footprint would be the
region where no cleavage is observed. So if a protein like a transcription factor
has flacked itself onto DNA the enzyme will not be able to see it the protein
will protect DNA from Nuclease digestion. Places where bands had been are
now absence when DNA binding protein binds. So Protein you were interested
in Bound to the particular sequence of DNA and protected it from digestion
leaving these footprints on the film.
Phylogenetic Footprinting: DNA recognition sites for regulatory proteins are
typically more conserved than the surrounding sequences. Regions that are
important are conserved throughout different species. Alignments with other
species where mutations are not tolerated are highly conserved. If a DNA
sequence is essential for some function any mutation for it will be quickly lost.
Regions that are highly conserved pop out at you , when you ask the computer
to align sequences, since it is in a Regulatory Region. Upstream is a good guess
that there is a Transcription Factor. You want to compare species that are
divergent enough so you dont have too many identical sequences so the
computer can point you to the happening regions. So DNA recognition sites for
regulatory proteins are typically more conserved then the surrounding
sequences.
Gel Shift Assays: The principle of this technique is that fragments of DNA with a
Bound protein move more slowly during gel electrophoresis than the same
DNA fragments without a bound protein. This is helpful to characterize the
specificity and avidity of the DNA-protein interaction. In robust, specific
interactions , the protein will specifically retard the electrophoretic mobility of
its target sequence. Very easy to see how protein binds to DNA sequence to see
how DNA runs through the gel. Can be done in ways that prove interaction is
specific and physiological relevant. Take small stretch of DNA that you believe
has Binding site for your transcription factor and make it Radioactive , if
protein is specifically bond to that DNA it will retard the mobility through the
gel. So if you change the DNA sequence it will not bind specific protein. The
protein will retard the mobility of the DNA IN THE Gel Electropheris
ChIP( Chromatin immunoprecipitation): Assay for protein binding sites on
DNA. This is used to identify the specific promoter regions that your
transcription factor occupies. This is a tool that allows us to see where
transcription factor goes in response to some stimulus . In response to some
signal some transcription factors turns on a whole flew of genes, get cell to
differentiate . You want to know which promoter the Transcription factor goes

too. Which genes get turn on by this particular Transcription factor. The protein
complex is recorvered, and then the occupied DNA is characterized in a variety
of ways including DNA sequencing, PCR amplification,or MicroArray anaylsis
. In this technology you have an Antibody that recognizes a particular protein
and when Antibody Antigen complex forms you can pull the complex out of
solution this is immunoprecipatation. Chromatin IP you have uninduced cells
and then you have cells that have been stimulated. In response to some stimulus
some transcription factor has began to occupy the regulatory region of small
subset of target genes , the genes that make muscle cells. You want to know
WHERE the transcription factor goes. After you have done induction and
Transcription factor have gone into Chromatin you add a cross-linking reagent
which is basicially formaldyde BRING TOGETHER molecules that are close to
each other inside the cell. When fix molecules DNAs and proteins that are close
to one another they become Insoluble and become a large mass. You crosslink
transcription factor to DNA, now you have to figure out which DNA
sequence!!!! So next thing you do is break DNA up into pieces use restriction
enzymes so you break DNA up into a lot of pieces. Some of the pieces will
contain Transcription Factor cross-linked to DNA or chromatin . Now that you
have broken it down into pieces you can have pretty good idea where
Transcription factor is. So after you need to use an Antibody that recognizes
your transcription factor and do an Immuno precipitation . Antibody that
recognizes Transcription factor these Antibodys have been linked to beads
(agrose beads) .Antibody bound to beads pieces of chromatin that contain
Transcription factors will bind to beads. Because Chromation is cross-linked to
Transcription Factors , the Transcription factor is the Antigen that is recognized
by the Antibody the Antibody is bound to beads. You throw in the beads with
the Antibody bound and you let it incubate and then you spin off the beads. So
you have beads bounds to Antibody with Transcription factor Chromatin
Complex . You can recover the Transcription factor Chromatin complez and
then reverse the crosslink. All you have left is pieces of DNA that correspond to
site where Transcription Factor bound. You can amplify the DNA using PCR or
use MicroArrays to find out where in the Genome they sit. If you use
Chromatin IP and then follow it with Micro Array its called Chip on Chip.
From Textbook definition: The DNA SITES that bind known transcription
factors in vivo can be determined by using ChIP , a transcription factor can be
localized to a specific promoter at a specific time. Proteins are covalently crosslinked to DNA with formaldehyde and then randomly sheared to yield
Chromatin fragments containing a few hundred bp of DNA. These chromatin
fragments are then immune-precipitated with Antibodies to a DNA Binding

protein and the enrichment of particular sequences is examined by quantitative

PCR or by hybrization to a microarray.

8.Describe the composition of the RNA pol II C-terminal domain and its
modifications after transcription initiation occurs. C-terminal Domain
site where a lot of Regulation occurs. In C-terminal domain of RNA polymerase
2 there is a repeat of seven Amino acids. Several: threonine, Serine, Tyrosine.
All of Hydroxyl group in their R group can be phosphorylated. Enzymes that
phosphorylate serine play a major role in regulating RNA Polymerase 2 helping
it go through the stages of Transcription. C terminal domain helps make sure
that all these things happen properly because there are kinases that recognize
these guys and phosphorylate them. There are proteins that will only bind to
Transcription Complex when Serine 2 is phosphorylated or when Serine 5 is
phosphorylated. CTD is a heptad repeat it has a lot of signaling potential. For
example enzymes that are involved in RNA processing at looking at the C
terminal domain they are watching the C terminal domain for the Green light
so they can do their next step. So capping, splicing, polyadenylation happen
along with transcription because it the MODIFICATIONS OF THE CTERMINAL DOMAIN of RNA POLYMERASE 2 that trigger these events.
RNA polymerase 2 is only there when Transciption is going on ,and RNA
polymerase 2 has a C-terminal tail hanging out, different residues become
phosphorylated and that summons the guys that do these other RNA processing
events. 5cap is addes when the message is fairly short but it has gone past
Initiation and gone to the Elongation phase. Capping Enzymes only come when
Serine 5 is phosphorylated during Initiation. Once intitation is done and Serine
5 has been phosphorylated then that is the signal for the capping guys to put cap
on 5 end of message. Once you added the Cap, then proteins that recognize the
cap start adding phosphates to Serine 2, when Serine 2 becomes phosphorylated
Serine 2 can only be phosphorylated once you add the 5 cap. The cap can only
be added once you phosphorylate Serine. Proteins that mediate splicing will
only come when Serine 2 and Serine 5 have been phosphorylated. That means
message is already being elongated and its already been capped splicing factors
then come. Serine 5 is dephosphorylated during elongation to enable the
switch to termination. So after Splicing is done Serine 5 becomes
dephosphorylated then you only have Serine 2 phosphorylated that is when it
switches from elongation to termination.
From Slide: Sequential
phosphorylation on Pol 2 CTD is involved in the switch from initiation to
elongation

1. Serine 5 is phosphorylated during initiation

2. mRNA capped by factors that recognize Ser 5-PO4
3. Cap binding proteins help recruit Serine 2 kinases to switch to elongation .
Serine 2 and 5 phosphorylated recruits mRNA splicing factors.
4, Serine 5 dephosphorylated during elongation to enable the switch to
termination.

9.Explain how the RNA polymerase II transition between initiation and

elongation occurs.
From initiation to elongation one of the events that happens is one of the
Transcription Factors that has Helicase- Kinase activity starts adding
phosphates to Serine 5 during Initiation , mRNA is capped by factors that
recognize Ser 5-PO4. Cap Binding proteins help recruit Serine 2 kinases to
switch to elongation. Ser 2/5 P04 recruits mRNA splicing factors. Serine 5 is
dephosphorylated during elongation to enable the switch to termination.
10.Explain how the RNA polymerase II C-terminal domain participates in
regulating different steps of transcription and RNA processing including
5cap, splicing, cleavage and poly-adenylation.
The C-terminal domain of the largest subunit of RNA polymerase 2 consist
of many copies of a seven amino acid repeat which undergo reversible
modification by phosphorylation. Immediately following transcription
initiation, the repeats are largely phosphorylated on the serine residue at
position 5. Capping Enzymes interacts with the Serine 5 phosphorylated
CTD. The cleavage and polyadenylation factors involved in 3 end
processing are recruited by interaction with the CTD phosphorylated at
serine 2.
After splicing is done Serine 5 becomes
dephosphorylated that is when is switches from elongation to
termination. Termination happens when you see this conserved
sequence, Conserved sequence near the end of the Transcript that
looks like AAUAAA THIS IS CALLED THE CONSENSUS SEQUENCE.
This conserved sequence AAUAAA in the message followed by a GU
rich region this the signal that you are getting near the end of Transcript
its time to cleave this transcript and add the poly A tail. Another example
of processing that is going on at the same time as Transcription. When
this AAUAAA is transcribed there are a series of proteins that recognize
that sequence the cleavage-stimulating factor and Cleavage and

PolyAdenylation specific factor. When they see those sequences, they

were originally attached to CTD they are waiting for that AAUAAA to
come out when they see it they leave RNA POLYMERASE 2 CTD and
jump onto to the transcript and they lead to cleavage and polyadenylation. C-terminal Domain of RNA Polymerase 2 is regulating
when and how splicing happens and poly-adenylation occur. Also
regulating capping and splicing of RNA. Once RNA has been cleaved
then there is an enzyme called Poly A-Polymerase that just adds a
whole stretch of As they are not encoded in the genome. Its just a
stretch of As that is added to the end of the message. There are proteins
that recognize Poly A tail these help stabilize the message also mark the
3 end of message and contribute to ways of translation of message.

11.Describe how different splice sites can be used to create multiple

mRNAs from a single gene.
You can make lots of different forms of the same gene product by picking
and choosing alternative EXONS.
Splicing is a mechanism carried out by snRNPs (RNA and snRNA) that
facilitate splicing there are particular RNAs that recognize Consensus
sequences that are present at two ends of Intron. 5Intron GU 3 Intron
AG. PLUS an A site called the branch point because of the role it plays
in splicing. This A is near the 3end. You have conserved sequences at
beginning of Intron 5GU and at end of Intron 3 AG.
So in many instances you have alternative processing where same primary
gene transcript can rise to multiple protein products, depending on which
exons are included or excluded. Decent amount of Human genome is
capable of giving rise to Multiple protein products. So again you can
have optional exons, Mutually exclusive exons, an exon in one transcript
and an intron in another transcript.
There are consensus nucleotide sequences that signal the beginning and end of
most introns. Introns can be short as 10nt, or as long as 100,000. Splice sites:
Are the sequences immediately surrounding the exon-intron boundaries The 5
splice site at the 5 end of intron includes the consensus sequence GU. The 3
Splice site at the 3 right end of the intron includes the consensus sequence AG.

Most genes are alternatively spliced. Exons can be picked and eliminated from a
Transcript. Average gene is able to give rise to 5 different mRNA transcripts
from same gene by picking and choosing alternative Exons. These multiple
protein products from the same gene are referred to as Isoforms. It contributes
to a variety of genes

Alternative Splicing is production of more than one mRNA and therefore more
than one protein product, from a gene by alternative exclusion of exons or
inclusions of introns from the mRNA.
Example : TropoMyosin: Is not a one size fits all thing cause the way
Actin/Myosin form is going to differ depending on the Contractile apparatus. It
would be great to have three different TropoMysoin molecules and you do but
they all come from same gene by alter native splicing.
12.Understand how usage of a splice sites can be regulated
Primary Transcript can convert into lots of different ways so that some
splice donors and acceptors are recognized and others are hidden. It is
all a function on how primary transcript has folded in three dimensional
space, which exons are visible to the splicing machinery, and which are
hidden. Splicing regulatory factors that allow the cell to make splice
choices that are appropriate for that cell type. Splicing and Regulation of
splicing is an Integral part of cells adopted or moving toward their
phenotype this way cells can make lots of different gene products from
the same gene depending on which splicing factors are present. Same
primary transcript can be acted upon in different ways as a function of
the cell type or other phenomenon going on inside the cell. Some
variations of them are Optional Exons or Optional Introns. Mutually
exclusive Exons. This is what you might see in tissue-specific gene
expression one exon specific for Muscle Cells or Skeletal Muscle cells.
Recognize Splice sites that are more readily accessible or visible. This is
a function on how primary RNA is folding who is sitting on it. Which
splice sites are seen by splicing machinery which splice sites are
ignored. Some splice sites are making themselves visible, and some are
obscured and splicing machinery is behaving accordingly. Generic
examples would be molecules that can interact with RNA and these
molecules can be RNA and protein the can act as repressors or
activators, they can obscure a splice site, for example you have a
Repressor that is sitting at the junction of the Intron-Exon junction so it

cant be recognized SO THIS SPLICE CHOICE WOULDNT BE MADE.

In another instance an activator might position itself in such a way to
make a particular Intron-Exon junction very readily visible to cell so it
would be chosen over other splice sites. There are lots of different
proteins that can bind to the primary transcript and affect the way its
folded and affect which splice sites are Recognize and which are not
recognize by the splicing machinery. Recognition of splice sites can be
influenced by RNA binding protein that can serve to make splice site
weaker or stronger. Splice sites are also regulated by the speed of
transcription If transcription is happing fast splicing machinery looks at
that message it will choose the Robust splice acceptor. If transcription is
happing slowly and there are lots of transcript that made the crappy
splice acceptor it will choose the crappy splice acceptor . So if
transcription is happening quickly you can have a strong splice acceptor
downstream of weak splice acceptor , but if transcription is happing fast ,
the time the splicing machinery gets there the better splice acceptor will
be visible and splicing machinery will choose better splice acceptor
whereas if cell is stressed, and slowing down its transcription that only
have weak splice acceptors visible and choose the weak splice
acceptor. SPEED OR RATE OF TRANSCRIPTION CAN DETERMINE
WHICH SPLICE SITE IS CHOOSEN!!!!!!!! Another thing that might
regulate splicing is the nature of the chromatin itself because
Nucleosomes affect rate of transcription and speed of transcription
which can affect splice site selection because if transcription is happing
slowly only the Upstream Splice acceptors is visible , Where as if
transcription is happing fast then both might be visible but the stronger
one might be chosen. OK if Histones are deacetylated and chromatin is
very condensed then there is a lot of nucleosomes packed this is going
to SLOW down the speed of Transcription and again if Transcription is
slower this affects which splice acceptors are visible to the splicing
machinery. Structure of Chromatin is affecting speed of Transcription,
which again is going to affect which splice sites are available for splicing
machinery.
Good Example:
Pyruvate Kinase has two alternate exons that can be chosen to give rise to
two different mRNAs that affects how robust the enzyme works it turns
out that factors that regulate these two alternate exons helps us
understand a key difference between Normal Cells and Cancer Cells.
Non cancer cells oxidize glucose completely to oxygen (Aerobic

Respiration get 36 ATPS from it. Cancer Cells do Aerobic Glycolysis

then typically followed by fermentation or Alcohol fermentation.
Glycolysis generates very little ATP. Only 2 ATP molecules compared to
36 ATP Reason Cancer cells just do Glycolysis is because cancer cells
are going through rounds of DNA Replication and Mitosis, growing in
number because they are dividing and proliferating constantly, they
need in addition to ATP they need organic material that they can use to
build the macromolecules of the cell, Nucleotides,proteins,lipids and
Carbohydrates . If you completely oxidize glucose all the way down to
Carbon dioxide you dont have any organic HydroCarbons molecules
that you can use to build lipids and everything else. Cancer cells do
Aerobic Glycolysis because the steps in breakdown of Glucose to
Pyruvate give the cell intermediates (Like Glyceraldehyde- 3 phosphate
that the cell can then turn into nucleotides or fats. How do cancer cells
get enough ATP ? they are constantly feeding on glucose they import
order of magnitudes more glucose then normal cells because they only
get 2 ATP from each glucose. Tumor cells are taking in order of
Magnitudes more glucose than normal cells they will show up in the
Body scan. Hot spots because cancer cells are dependent on so much
more glucose. Tumor Cells take Glycolytic intermediates along the way
as much as they need ATP they also need organic compounds that they
can use to build other Macromolecules, so they can grow very quickly. In
cancer cells the Pyruvate doesnt go into Mitochondria and do Krebs
cycle, Electron Transport. ALTERNATIVE SPLICING: The enzyme
Pyruvate Kinase, can be spliced in two different ways. One way makes it
very active and pyruvate goes directly into the Mitochondria for Krebs
Cycle. The other splice variant is pathetic very slow at getting Pyruvate
into Mitochondria this slows down whole Glycolytic pathway that allows
intermediates between Glucose and Pyruvate to accumulate, this allows
the intermediates to be shunted off, to other Anabolic pathways inside
the cell to build other macromolecules . Switching from one ISOFORM
to the other is done by Two alternative exons withen the same enzyme,
one gives you a robust enzyme and one exons gives you a pathetic
enzyme. This evolved becaue there are situations where you want
deliberately slow down Glycolysis. So all intermediates are shunted off
for other purposes. Why do cancer cells choose the crappy exon over
the ROBUST exon? It all has to do with which of these hnRNP binding
proteins are present in the cell. Alternative Splicing factors are the result
of other alterations inside the cell because whatever is driving
expression of those splicing factors has been deregulated as well as

more of a consequence. So IF YOU can get into this system and pick up
Robust Exons so you can slow down the rate at which cancer cells
grow. Binding of the hnRNPS to the splice sites flanking exon 9 in PKM
transcripts results in exon 9 exclusion and exon 10 inclusion generating
PKM2 this converts PEP to pyruvate less efficienctly then PKM1 leading
to accumulation of glycolytic metabolites for anabolic metabolism. The
reason that the splicing choice changes is because a number of
Transcription factors that are typically overexpress in cells are affecting
the guys that regulate splicing . N-Myc and E2F1 are Transcription
factors that allows cells to enter S-phase (which is DNA
Replication(Normal cells arrest their Cell Cycle progression they come
out of Mitosis go to G1 and then Stop. They need a strong acting force
that allows them to enter another cycle of DNA replication. N-Myc and
E2F1 allows cells to get over this hurdle these transcription factors are
typically deregulated meaning there is too much of them or
OVERACTIVE. N-Myc and E2F1 are juiced in Cancer cells that would
explain Transcription factors associated with Stem cells. Cancer cells
tend to behave like Stem cells that are more proliferative. STAT3 is
another family of Transcription factors. These are Transcription factors
that are not acting normally in Cancer cells.

13.Understand RNAi and the implications on gene expression.

Small Regulatory RNAs or RNA silencing or RNAi additional way of
controlling gene expression, Generating RNA molecules that are
complementary to mRNAs they can base pair with mRNA, base pairing
with mRNA can either target it for degradation or inhibit its translation or
both. Small RNAs that are regulating activity of mRNA this is a whole
other half to gene expression, in addition to the genes encoding
positively acting messages that makes proteins (Luke Skywalker). The
light saber battle is between mRNA and these interfering RNAs that are
also encoded in the Genome. Encoded in the Genome make short RNA
molecules that antagonize mRNA THEY ARE DARTH VADERS short or
20 or so Nucleotides long. But they can specifically base pair with a
mRNA and target it for degradation or interfere with its Translation. So if
you have ridiculous amounts of mRNA and you also have interfering
RNA YOU will not get any protein. These small RNAs were the Darth
Vader to Luke Skywalker and they were affecting regulation of gene

expression in profound ways. In research it allows to experimentally

silence a gene that you are interested in. It also leads to lots of therapy
for disease such as cancer. Example
C.Elegans worms expressing a GFP fashion gene GFP Reporter gene.
When cells express it they make Green Fluorescent protein fused to
whatever it what fused to. You can make C.Elegans fluorescent nice and
green. If you put this interfering RNA against GFP then worms stop
being green. Interfering RNA show complementary version of GFP
transcript shut down GFP transcript. So the phenomenon of having a
short RNA molecule that is capable of inhibiting translation of preexisting mRNA is given the general name RNAi : Meaning you are
interfering with Translation of message . So remember RNA that is
doing interfering can come from the cell itself, our genomes encodes
400 different interfering RNAs they effect expression of 1/3 of our genes.
A lot of our genes are regulated by the level of interfering RNA that
messes with our message and interferes with Translation. From
slide : Small RNAs have regulatory functions, including shutting down or
altering gene expression. Previously, small RNAs had been ignored,
while scientists focused on full-length mRNAs.
PETUNIA
EXPERIMENT IN 1990!!!! Didnt get purple flowers got white
flowers.
These small RNAS that use RNA-RNA base pairing identify mRNA targets
for regulation. These are versatile in regulatory outcomes they produce,
but control translation or stability, dependent on the mRNA identity and
extent of base pairing.

Micro RNAs : Still small RNAs Animal and plant genomes code for many
short RNA molecules called microRNAs. For example Humans make
about 400 of these, and they control the expression about 1/3 of our
genes. The targeted mRNAs have conserved sequences, usually in their
5 to 3 untranslated regions. Known mi-RNA regulated events include
:Mammalian immune system, apoptosis and cell proliferation in fruit flies.
Leaf and flower development in plants Neuronal asymmetry in worms,
BloodCell differentiation in humans, tissue specific gene expression in
humans.
We can manipulate cells by introducing small
oligonucleotides and down regulate gene expression on our own.
Laboratory generated or cell generated all different ways of regulated

gene expression. Basic tool for regulated gene expression that evolved
a long time ago it is present in many organisms like worms and flies.
Has been maintained is a whole other way of regulated gene
expression. In the genome, transcribing gene that encodes micro-RNA
makes Micro RNA. The micro-RNA then needs to be processed down
to generate a small double stranded RNA that is folded over on itself ,
There is a whole biochemical pathway that makes this happen. There
are enymes that recognize this short micro-RNA and trim it. Eventually it
gets to point where two strands are separated from each other and
somehow there is a protein complex that knows which strand is the
interfering RNA that is complementary to the message. When it binds
the message what happens when this double stranded part of the
message is seen by the cell it targets it for DEGRADATION or stalls the
movement of the Ribosome or it stalls protein synthesis. The message is
either degraded or chewed up or not degraded but it then cant be
translated easily because interfering RNA are sticking on end of
message. Ribosomes are being stall because of this complex. These
microRNAs either repress translation or they promote degradation. But
either way they are interfering with expression of gene. This may
have evolved as a defense mechanism against viruses. Some viruses
have a double stranded RNA genome, Viruses are good for cells so
cells have evolved a way to recognize double stranded RNA and see it
as something that needs to be eliminated. So Viruses typically present
themselves as double stranded RNA . So viral DNA comes in Double
Stranded and specificailly down regulate a particular gene by introducing
a short double stranded RNA where one the strands is complementary
to the message. Double stranded RNA is recognized by components
of the RNA machinery which then separtes the strands and presents the
one that is complementary to the message to the message in such a
way that interferes with Translation promotes Message degradation. So
in addition to interference with Translation of Messages in the
Cytoplasm these interfering RNAs can also play a role in shutting down
gene expression by affecting the structure of chromatin they can affect
the ability of chromatin to be read or not be read. IMPORTANT THING
TO REMEMBER IS INTERFERING RNA do more then mess with
message translation they also play a role in Chromatin and
heterochromatin state, Thus regulate gene expression
A lot of Micro RNAs are expressed in a Tissue-Specific fashion one is
expressed in Neural Tissue, skeletal muscle ,liver . Tissue Specific

regulation is also going to affect gene expression in a tissue specific

way. If you feel a particular disease is the result of the overexpression of
a particular gene ? You can prove this by introducing an RNAi, down
regulate that gene and if the disease phenotype goes away. You can
use interfering RNA in therapy. When scientists use small RNAs they
can down regulate an gene and reverse a disease phenotype. miRNAs:
are though to play a regulatory role in development, patterning of the
nervous system, control cell proliferation and cell death, leaf and flower
development in plants, differentiation of various cell types , there are role
of microRNAS in the development of cancer.
14.Explain the composition of the ribosome and how it orchestrates tRNA
and mRNA to synthesize polypeptides.
Ribosome is made up of RNA and proteins. Ribosome is the structure that
mRNA is going to be tunneling through ,it is going allow for the message
to be read. The Ribosome is Catalytically active it is forming peptide
bonds between amino acids as they come along. Ribosome is made up
of Ribonucleic protein. Prokaryotic and Eukaryotic Ribosomes are
slightly different from one another but our overall 2 structure is similar
like our sequences are different but the overall structure are conserved
especially the surfaces that bind or interact with tRNA, mRNA, proteins.
The size of the Ribosome itself, the small and large subunit and RNAs
are all given with the coefficient of S. which has to do with
sedimentation. Eukaryotic Ribosomes are larger then prokaryotic
ribosomes but Ribosomes have a small and large subunit. In eukaryotes
40S AND 60S. IN PROKARYOTES 30S 50S. Each subunit has one ot
two RNAS. The small subunit in prokaryotes has 16s RNA AND IN
EUKARYOTES IT IS 18S. Each protein is typically present in one copy.
Important in Eukaryotes Ribosomal proteins and Ribosomal RNAs have
to find each other to build the Ribosome. Eukaryotes have a special
organelle call the Nucleolus. Nucleolus is found inside the nucleus.
Nucleolus is the site of assembly or partial assembly of the Ribosome.
Ribosomal RNA and Ribosomal proteins partially assemble themselves
into the Ribosome in the Nucleolus and then they go into the Cytoplasm
where they finish. Using GFP showing that there is green staining in the
Nucleolus because Ribosomal protein is made in the Cytoplasm of older
Ribosomes and then they go into the Nucleolus where they join with
Ribosomal RNA and begin this phenomenal task of assembling into the
Ribosome part if it happens in the Nucleolus and part of it happens in

the Cytoplasm.
Christmas Tree Appearance: IS showing you very active transcription
of this clusters of genes encoding Ribosomal RNAs.
The Cap and poly A tail are cues for Translation factor binding as well as
nucleases. Intitation factors are going to grab the message, Recognize
Cap and poly A binding proteins and put the message in a conformation
that the Ribosome can see, that the translation Complex can see.
Ribosome needs to see where the cap is needs to see the poly A tail
these factors put the message in a good conformation that will make the
Ribosome ready to jump on it and start translating. You have to
assemble whole transcription complex , the message, the tRNA , the
small ribosomal subunit ,large ribosomal subunit, Once this all set , its
just banging out one codon at a time. Small Ribosomal subunit and
tRNA recognize AUG are the first things that happen then that will lead
to the formation of the whole Ribosome. First cell needs to know where
it is going to start. And once that is good to go, it will continue from
there. Once you have initiation, once Ribosome has found AUG and
tRNA that encodes Methione has found AUG then you are set to go and
the rest of Ribosomes comes on board, The large subunit of Ribosome
joins and then Initiation is over Remember Pre-Initation Complex: eIF(a
G-protein/GTP) met tRNA and small ribosomal subunit. The Cap
recognition complex targets mRNA to pre-Initiation complex. Ribosome
scans mRNA for init AUG. when met tRNA binds AUG ,eIF GTP
hydrolysis leads to dissociation of eIF 2a dissociation and large
ribosomal subunit binds. The Elongation of Translation consists of a
series of three step cycles as each amino acid is added to the
proceeding one. 1. Codon Recognition = Binding of charged tRNA to
ribosome A site (hydrogen bonding between codon and Anticodon)
A=Aminoacyl. 2. Formation of peptide bond , During peptide bond
formation, an rRNA molecule catalyzes the formation of a peptide bond
between the polypeptide in the P site(P=peptidyl_ with the new amino
acid in the A site (Note that the linking occurs through the carboxyl end
of growing chain to amino group of an amino acid on amino acyl tRNA.)
3. Translocation: The ribosome moves along the mRNA, one codon at a
time. Ribosome moves in 5 to 3 direction, leaving a vacant A site for
the incoming charge tRNA. The Ribosome can thought to have three
sites, tunnels ,Cavities where the codon are visible. These sites are
referred to as A site P site E site. What the point of Ribosome is doing is
moving down the message and as each new codon comes into view its

is asking for the amino acid that codon specifies to join the growing
peptide. We have a tRNA that is sitting in the peptide site and it is
holding the growing protein it is now ready to request Amino ACID 5
The codon requesting Amino acid 5 is in the A site for Amino acid , the
first step is codon recognition that means the tRNA whose Anticodon
loop can base pair with this codon has to snuggle into this site. Only
tRNAs that base pair and are stable can stay the. So when rite tRNA sits
in the A site that is codon recognition now the amino acid attacks
growing polypeptide in the Psite so a new covalent bond is formed. A
covalent bond forms between growing peptide that is being held by
tRNA in p-site and incoming amino acid in the A site. Once peptide bond
formation has happen we dont need P-site tRNA and we are ready for a
new codon to be read. The next step that has to happen is translocation
, Ribosome needs to move along message so a new codon comes into
view. When Ribosome translocates or moves along the message the
tRNA that was in the P-site goes into the E site . tRNA that was in A site
is now in the P-site because it has the peptide and now codon is ready.
15.Explain how an amino acid becomes attached to its tRNA.
tRNA function as adaptors in reading information from RNA into synthesis
of protein. Molecules of tRNA are not all identical, each carries a specific
amino acid on one end and each has an Anti-Codon on the other end.
Each tRNA is used repeatedly to pick up its designated amino acid in
the Cytosol, to deposit the amino acid at the ribosome and to return to
the Cytosol to pick up another copy of that amino acid. Transfer RNA
genes are located in small clusters scattered around the genome. tRNAs
have promoter sequences within the coding region of the gene
(transcribed by RNA pol 3) During processing, the tRNA precursor is
trimmed and numerous bases must be modified. The reaction in which
an amino acid is attached to a tRNA is catalyzed by aminoacyl-tRNA
synthase. The specificity of the genetic code is mediated by the
codon/anti-codon interaction. However a tRNA that binds to an mRNA
codon specifying an particular amino acid must carry only that amino
acid . There fore the accuracy of the aminoacyl tRNA synthase is
attaching the correct amino acid to the correct tRNA is crucial. There is
20 different amino acids and therefore there are 20 different synthetases
that match the 20 different amino acids So charging of tRNA means it is
holding the amino acid its specific for. All tRNAs have a characteristic LShape with the Amino Acid attached at the 3 end, the anticodon loop is
attached to the opposing end of the molecule CCA common to 3

terminus to all tRNAs. Charging of tRNA with correct amino acid is

carried out by Aminoacyl-tRNA synthase.
16.Understand the key events during (a) initiation of translation, (b)
elongation of a polypeptide, and (c) termination of protein synthesis.
a. Initiation of Translation: So small ribosomal Subunits binds to mRNA.
An initiator tRNA met , with anti-codon 3-UAC-5 base pairs with AUG
start codon. This binding determines where the new protein will begin ,
and sets the reading frame. In eukaryotes, proteins bound to the 57mpg
cap are recognized by the small ribosomal subunit, which scans along
until the first AUG is found. Cap recognition complex targets mRNA to
pre-initiation complex. Ribosomes scans mRNA for intitation AUG when
met tRNA binds AUG,eIF Hydrolysis which leads to eIF dissociation,
large subunit binds.
b. Elongation of Translation: Consists of a series of three step
cycles as each amino acid is added to the proceeding one.
1. Codon Recognition: Binding of a charged tRNA to ribosome A site
(Hydrogen Binding between Codon and Anti-codon)
2. Formation of Peptide Bond: During peptide bond formation, an rRNA
molecule catalyzes the formation of a peptide bond between the
polypeptide in the P SITE with the new amino acid in the A site (Note
that linking occurs through the Carboxy end of growing chain to
amino group of amino acid on amino acyl tRNA)
3. Translocation The ribosome moves along the mRNA, one codon at
a time. Ribosome moves in 5 to 3 direction leaving a vacant A site
for the incoming charged tRNA. Translocation is fueled by the
hydrolysis of GTP. Effectivlely Translocation ensure that mRNA is
read in 5 to 3 codon by codon.
Termination of Protein Synthesis: Stop Codon into A site. Release
factor eRF1 binds, promotes hydrolysis of peptidyl tRNA aa bond.
Protein is released, everyone dissociates. UAA,UAG,UGA codons signal
translation stop. These codons are not recognized by tRNA, but by
release factors. The releae factor causes the addition of a water
molecule instead of an amino acid. This reaction release the
polypeptide, and triggers the disassembly of Ribosomes into subunits.
Binding of release factor instead if tRNA to the A site triggers Hydrolysis
Reaction cleaves the growing polypeptide from tRNA in the P-SITE and
everything comes apart.
17.Understand the principle of posttranslational protein targeting

Most proteins are synthesized in the Cytoplasm, targeting signals built into
the polypeptide chain direct proteins to various cellular compartments
including the nucleus, endoplasmic reticulum, mitochondria, chloroplasts
and peroxisome. Targeting to the Endoplasmic Reticulum occurs
DURING TRANSLATION. POST translational targeting directs proteins
to other organelles. Targeting sequences are necessary and sufficient to
specific organelle. The targeting sequences may remain as part of the
protein, or they may be cleaved. Permanent targeting sequences may
allow a protein to continue to transit from one place to another. Some
proteins have sequential targeting sequences (more then one target
sequence) targeting sequences are recognized by receptors or escorts,
or they cross channels called Translocons.
What is a Translocon ?
Membrane- Bound Protein Translocator systems (Translocons ) move
specific proteins across membranes in an unfolded conformation.
Sequences withen the transported proteins are recognized by
translocons. Translocons are found in all three domains so they must be
ancestral. For example the Sec translocons direct proteins in to the
endoplasmic reticulum in eukaryotes and out of the cell in prokaryotes.
Signal sequence in protein acts a targeting mechanism to deliver to proper
organelle.
18.Know what chaperones are and how they work
Chaperones use energy of ATP to help proteins from aggregating and to
help them move.
So remember the Cytosol of the cell is aqueous. Family of proteins that
coat or cover the Hydrophobic stretches to prevent the protein from
forming a hopelessly useless mess of aggregated Hydrophobic amino
acids.
Also Heat shock proteins cover or coat that Hydrophobic stretches and
prevent proteins from inapprotaitly aggregating with one another. Heat
shock proteins is what gets turn on when cell gets blund up with a whole
bunch of misfolded proteins. Heat shock proteins bind to Hydrophobic
stretches and use energy of ATP to help protein get itself into a betterfolded state. Chaperones dont fold proteins, but by binding hydrophobic
amino acids they inhibit aggregation. There are specialized chaperones
for some proteins these are generic. Chaporonins aid in import process
become associated with protein while still in the cytoplasm, association
requires energy from ATP. Chapornins aid in unfolding proteins so it can

travel through organelle membrane.

19.Know what translocons are
Translocon are membrane-bound Protein Trans locator systems which
move specific proteins across membranes in an unfolded conformation.
Sequences within the transported proteins are recognized by
Translocon. Translocons are found in all 3 domains so they must be
ancestral. Example Sec Translocons direct proteins into the
endoplasmic reticulum in eukaryotes and out of the cell in prokaryotes.
Characteristics of protein translocation: Amino terminal targeting signal
or transit sequences. Some sequences are cleaved some are not.
Hetero-oligomeric trans membrane channel that is gated across AND
within the plane of the membrane. Peripherally-attached protein
translocation motor powered by ATP or GTP Hydrolysis( this is
REQUIRED to pull this protein across the membrane and make sure
movement is directional. Topogenic sequences on protein for membrane
insertion for proteins destined to be inserted into a membrane rather
than cross it.
Motor may be a chaperone, a protein folding enzyme,heat or cold shock
proteins, or a ribosome in the case of co-translational translocation.

20.Know the 4 possible destinations of mitochondrial proteins and the 6

possible destinations of chloroplast proteins
Mitochondria have four possible destinations for proteins:
1.) outer membrane,(Is Fairly smooth) 2.)Inter-membrane space, 3.)Inner
membrane,(Is very convoluted, enzymes for electron transport are there)
The more surface area you have the more ATP you generate.
4.) matrix
But remember 95% of mito proteins are nuclear encoded. Mitochondra has
only 37 genes. In the Mitochondria the whole translocon is called the
TOM-TIM COMPLEX.
TOM: Translocon Outer membrane Mitochondrial Membrane
TIC: Translocon Inner Mitochondrial Membrane
There is no such thing as a Translocon that is big enough to fit an folded
protein. The chaporones or HSP proteins are helping the protein unfold
and keeping from aggregating on itself because they are sticking to the

Hydrophobic portions. So there is a lot of choices for protein it is based

on its signal sequence and the fellows that recognize the signal
sequences to make sure the protein gets to where its needs to be. In
Inner mitochondrial Membrane there are two TIM complexes there is
one that goes all the way through to send the protein to the matrix and
the ones with the side door that allow the protein to come out the side, if
protein needs to remain within the Membranes.
Importing sequences into the mitochondria: The polypeptide is targeted to a
mitochondrion by a targeting sequence, which is located at the Nterminus in the matrix protein and is located internally in most inner
membrane proteins. Cytoslic HSP 70 molecules unfold the polypeptides
prior to entry into the mitochondrion. The proteins are recognized by
membrane receptors and trans located through the Outer Mitochondrial
Membrane by way of pores in the TOM Complex of the Outer
Mitochondrial Membrane .
Most intergral proteins of the Inner Mitochondrail Membrane are directed to
the TIM 22 complex of the Inner Mitochondral membrane which steers
them into the lipid bilayer of IMM. Mitochondrial Matrix proteins are trans
located through the TIM23 complex of the IMM. Once the protein enters
the Matrix, it is bound by a Mitochondrail Chaperone which may pulls the
polypeptide into the matrix. Once in the matrix, the unfolded protein
assumes its native conformation with the help of HSP 60 chaperones.
Chloroplasts have six possible destinations for proteins:
1.
2.
3.
4.
5.
6.

Outer Membrane
Inter-Membrane Space
Inner Membrane
Stroma( Aqueous environment inside the Chlorplast)
Thylakoid Membrane(Photosynthetic pigments are here)
Thylakoid Lumen
The chloroplast import Mechanism is similar to mitochondrial, but the
proteins are not shared. The chloroplast import proteins are most
closely related to cyanobacteria proteins involved in secretion, which
makes sense given ancestral relationship. N-terminal transit
sequences are necessary and sufficient but not defined. They vary in
length but seem to be rich in serine and threonine , and have a net
positive charge. The chloroplast targeting sequences are not as well
defined as Mitochondrial Targeting sequences. Inside the Chloroplast
there is another membranous structure called the Thylakoid. Space
inside the Thylakoid is called the Thylakoid Lumen. Again there are

compartments of the Translocon that can help target proteins to

various Chloroplast Membranes. And if proteins need to go into
Thylakoids they would get sequenced for that as well. As we saw in
Mitochondria there are proteins that are synthesized inside the
chloroplast, the chloroplast also has its own genomic DNA its own
Ribosomes tRNAs so proteins that are synthesized inside chloroplast
need to be targeted as well have their own translocon system.
21.Know the similarities and differences between mitochondrial and
chloroplast translocon systems.
Chloroplast has TOC AND TIC COMPLEX
Translocon outer Chlorplast
Tranlocon Inner Chlorplast
Again in Chlorplast proteins are coated with Chaporone proteins that keep
them unfolded so they can they pass through the Tranlocons. They are
passing through outer and inner Chloroplast Membrane, inside the
Chloroplast is the aqueous compartment the Stroma or additional set o
membranes, where photosynthetic pigments are that are called
thylakoids. If protein is just crossing Inner and Outer Chloroplast
Membranes and ending up in Stroma it can then just begin folding but if
it is going to thylakoid membrane or thylakoid lumen it has more
targeting to do. Again there are components of the Translocon that can
help target proteins to various Chloroplast Membranes and if they need
to go to Thylakoid they would get targeted for that as well. Proteins
coming from Cytosol of cell crossing TOC and TIC once inside in the
Stroma it can go Thylakoid membrane or they can become part of the
Thylakoid Lumen. (Site of photosynthesis) Both mitochondria have
sequences that help protein to get to various locations. Proteins that
need to cross the Thylakoid Lumen have a secondary N-terminal
sequence and cross the thylakoid lumen by a special translocon.
Proteins destined for the Stroma contain a stroma targeting domain at
their N-terminus , whereas protein destined for thylakoid contain both a
stoma targeting domain and a thylakoid-transfer domain at their Nterminus. Stromal proteins remain in the stroma following translocation
through the outer envelope and removal of their single targeting
sequence. The presence of the thylakoid transfer domain causes
thylakoid proteins to be trans located either into or completely through
the thylakoid membrane. A number of proteins of the thylakoid

membrane are encoded by chloroplast genes and synthesized by

chloroplast ribosomes that are bound to the outer surface of the
Thylakoid membrane Those that need to enter the thylakoid membrane
by a special translocon.
22.Know the structure and functions of mitochondria, chloroplasts and
peroxisomes.
Know shared and unique properties of these organelles.
Mitochondria: Oxidative phosphorylation, TCA cycle, fatty acid oxidation,
amino acid and Nitrogen metabolism. Steroid Metabolism,porphyrin and
heme synthesis, regulation of programmed cell death, regulation of insulin
secretion, thermogenesis,DNA,RNA,and protein synthesis.
Chloroplast:
Photophosphorylation,CO2
reduction,photorespiration,
chlorophyll synthesis,lipid synthesis,DNA,RNA, and protein synthesis.
Peroxisomes: B oxidation of long chain fatty acids, detoxification of
Hydrogen peroxide, alcohol oxidation, synthesis of plasmalogens.
In Eukaryotes Aerobic Respiration takes place in the Mitochondra.
Mitochondrai arose by fusion of a bacterial cell and an archaeal cell, or
symbiosis with primitive eukaryotic cell. All known mitochondria retain some
bacteria-derived genes. The Human mitochondrial genome is circular and
contains 37 genes: encoding 13 mitochondrial proteins, 2 mitochondrial
ribosomal RNAs and 22 mitochondrial tRNAs . Mitochondrial RNA
polymerase mitochondrial ribosomal proteins, and even the enzyme of the
Citric Acid Cycle are encoded in the Nucleus. Over 600 Mitochondrial
proteins synthesized in the cytoplasm. Mitochondrail genes on
Mitochondrail DNA: 13 Mitochondrial Membrane Proteins, 22tRNAs 2
rRNAs . Typical Mitochondria are bean-shaped organelles but may be
round or threadlike. Size and number of Mitochondria reflect the energy
requirement of the cell. The way Mitochondria reproduce also provides
evidence of their prokaryotic ancestry. Mitochondrial fission is very similar
to the bacterial process. Both fusion and fission are regulated by GTPases.
Defects in these processes are associated with neuromuscular
diseases.Mitochondra play a role in determining the food oxidizes is going
to be used to generate ATP or Heat(Thermogenesis). Hibernated
organisms combust glucose more to make heat, to make ATP because
they are not moving that much. Mitochondria are capable of replicating their
DNA of transcribing their DNA into RNA and translating their mRNA into
protein. Translocons are doing the work of getting all the enzymes that
mitochondria needs and lots of Ribosomal proteins all of these have to
pass through Translocons for Mitochondria to function. Mitochondrial

genome is in the Matrix of the Mitochondria. INNER MEMBRANE OF

MITOCHONDRIA is the site of ATP Synthesis and energy generation. The
components of the Endoplasmic Reticulum play a major role in
Mitochondrial constriction and splitting.
Mitochondrial Structure and Function: Mitochondria have bean-shaped
structure because of all of its Membraneous fold. The folds of the inner
membrane are called Crista. The in-foldings of the Inner Mitochondrial
Membrane create more surface area for all Metabolic activitys that are
associated with Inner Mitochondrial membrane. The Outer Mitochondrial
Membrane is somewhat porous they have a lot of these large and loose
pores these Beta Barrall proteins that are fairly accommodating and can
allow things to enter they are Reminscenet of Bacterial Proteins, they make
it easy for molecules to cross Outer Mitochondrial Membrane. The Inner
Membrane of the Mitochondria is very impermeable even to protons it has
different kinds of lipids associated with it.
Chloroplast Structure and Function: PhotoSynthesis takes place in the
Chloroplast, Chloroplast are very similar to Mitochondria in which they have
these dense membranous structures where enzymes are situated or
Electron transport chain is situated . The chloroplasts are a lot bigger then
Mitochondria it is similar to Mitochondria in which it has it own genome it
can replicate its own DNA and it can synthesize its own proteins . Also
Chlroplast have A LOT more genes then mitochondria Chlorplast has about
250 genes . Chloroplast like Mitochondria has a double Membrane outside
of it , there is outer,inter and inner membrane the stuff inside inner
membrane which is Mitochondria was called the Matrix , In the chloroplast
it is called the stroma. In the chloroplast it has a whole other set of
membranes inside them those are called the Thylakoids(space inside the
Thlykoid space or Thylakoid Lumen. In chlrosplast there are six different
places where proteins can go. They can go to outer membrane,
intermembrane space ,inner membrane, stroma , thylakoid membrane and
thylakoid lumen. Chloroplast are larger then Mitochondria and have 3
membranes. The chloroplast stroma like the mitochondrial matrix contains
DNA and ribosomes . In chloroplast there are 6 different places proteins
can go as opposed to the mitochondria where there is only four places.
Chloroplast do photosynthesis which utilizes reaction centers , which are
transmembrane complexes of proteins,pigements, and redox cofactors that
absorb light , capture high energy electrons that can be used to generate
proton gradients produce ATP. Photosyntheis requires the thylakoid
membrane and the stroma. Two sites of photosynthesis Thylakoid and
Stroma. So you have photosynthetic pigments which are molecules that

can absorb certain wavelength of light and become excited by them.

Electrons in photosynthetic pigment become excited by certain wavelength
of light and when they are excited they can donate high energy electrons to
processes that eventually give rise to ATP and NADPH. PhotoSynthesis:
Involves the photo part where photosynthetic pigments embedded in
Thylakoid Membranes become excited by photons of light that excitation is
used to power electron transport chains which are used to make ATP. ATP
here is made by photophosphorylation as opposed to oxidative
phosphorylation. It is the same idea electron transport generated proton
gradient which powers ATP Synthase. ATP Synthase in chloroplast is the
same as ATP Synthase in Mitochondria.
1st part of Photosynthesis: High energy electrons excited by light are used
to make ATP and NADPH this take place in Thylakoids
2st part of PhotoSynthesis: Synthesis part takes place in the Stoma liquidty
part this is where enzymes from Calvin Cycle in which they take inorganic
Carbon and fix it (reduce it and turn it into Organic Molecules which then
be turned into sugars and amino acids. This all takes place in the Stroma ,
this requires CO2 which diffuses into the plant this is called the DARK
REACTIONs they dont need light but they require ATP and NADPH.
Peroxisomes: B oxidation of long chain fatty acids, detoxification of
hydrogen peroxide, alcohol oxidation synthesis of Plasminogen.
Peroxisomes differ from mitochondria and chloroplasts in many ways. Most
notably, they are surrounded by only a single membrane, and they do not
contain DNA or ribosomes. Like mitochondria and chloroplast , however
peroxisomes are thought to acquire their proteins by selective import from
the Cytosol.
Textbook Definition of Eukaryotes: Membrane bound
organelle containing oxidative enzymes. Peroxisomes seem to have a
Unique targeting sequence that allows proteins to get in there. There are
short amino terminal sequences, Carboxyl terminal sequences that are
found in proteins that target them to the peroxisomes. (A specific sequence
of three amino acids, called PTS1 located at C TERMINUS OF MANY
PEROXISOMAL PROTEINS functions as an import signal. Other peroxisomal proteins contain a signal sequence near the N TERMINUS called
PTS2 . If either of those sequences is experimentally attached to a
cytosolic protein, the proteins is imported into the peroxisomes. The import
procress is still poorly understood, although it is known to involve soluble
receptor proteins in the cytosol that recognize targeting signals, as well as
docking proteins on the cytosolic surface of the peroxisome. Peroxisomes
have some anabolic and catabolic activities primarily their breaking down
activity because they like to break down molecules using oxygen.

Peroxisomes are found in all eukaryotic cells. They contain oxidative

enzymes, such as catalase . Like mitochondria peroxisomes are major
sites of oxygen utilization. One hypotheis is that peroxisomes are vestigae
if an ancient organelle that performed all the oxygen metabolism in the
primitive ancestors of eucaryotiv cells. Peroxisomes might have served to
lower the intracellular concentration of oxygen , while exploting its chemical
reactivity to perfrom useful oxidative reactions. The later development of
mitochondria rendered peroxisomes obsolete, because many of the same
reactions which had formerly been carried out in peroxisomes without
producing energy were now coupled to ATP formation by means of
oxidative phosphorylation. Peroxisomes are membrane bound vesicles that
contain oxidative enzymes. Peroxisomes oxidize very long chain fatty acids
to Acetyl CoA , hydrogen peroxide (H2o2) a reactive and toxic compound is
formed in peroxisomes and is broken down by the enzyme catalase. A
major function of oxidative reactions performed in peroxisomes is the
breakdown of fatty acids molecules. In a process called B oxidation, the
alkyl chains of fatty acids converted to acetyl coA. An ESSENTIAL
biosynthetic function of animal peroxisomes is to catalyze the first reactions
in the formation of plasmalogens, which are the most abundant class of
phospholipids in myelin. Deficiency in plasmalogens causes profound
abnormalities in the myelination of nerve cells, which is one reason why
many peroxisomal disorders lead to neurological disease. Peroxisomes are
called this because they have the enzymes that break down peroxide , they
can use oxygen from atmosphere to break down organic molecules which
generate peroxide. Peroxisomes look very dense inside(that is because
they are packed with so many enzymes, they are very dense and rich in
Enzymes.

23.Understand how mitochondria generate ATP (the electron transport

chain and ATP synthase)
Mitochondria is the place where most of the energy is recovered, because
end stages of oxidation of food take place in Mitochondria. Our bodies are
able to recover energy from proteins,Carboydrates,fats . Energy that is
recovered from these C-H bonds is used to make ATP. That takes place
deep inside the Mitochondria of the Cell. End stages of Metabolism take
place in the Mitochondria . That is where all the energy striped down from
these organic molecules is used to make ATP. Carbohydrates and fats are
broken down in the cytoplasm of the cell initially and then final stages of
oxidation take place inside the Mitochondria . They produce Acetyl CoA

which goes into the Krebs Cycle and the energy rich molecules that result
from it are used to make ATP . Pyruvate gets converted to Acetly CoA in
the Mitochondria. Acetyl CoA is made from oxidation of fats and glucose
goes trhough Kreb Cycle which takes place in the Mitochondrial matrix. So
Carbons are completely oxidized ,energy is held in these energy rich
molecules e carriers NADH and FADH2. The Citric Acid cycle in the
Mitochondrial Matrix catalyzes the complete oxidation of the Carbon atoms
in acetyl coA . Kreb cycle converts Acetyl CoA to carbon dioxide and all of
the high energy electrons are in the form of reduced electron carriers
NADH and FADH2 . The next step is to use these high-energy electrons to
produce ATP. The end of oxidation of food is that organic molecules are
turn into inorganic molecules. High energy electrons that were associated
with these organic molecules are given to energy carriers or electron
shuttler molecules. OK SO THE INNER MITOCHONDRIAL MEMBRANE IS
WHERE THE CELL TO MAKE ATP USES THE HIGH ENERGY
ELECTRONS. Electron carriers that are now going to donate those
electrons to something called the Electron Transport Chain which are a
series of molecules that are embedded in Inner Mitochondrial Membrane.
Each one is more Electronegative to one the next to it. Each one is able to
accept electrons from the one next to it become reduced and donate the
electron to one after it. Electrons can only move in one direction. They are
moving from a molecule that is less electronegative to a molecules that is
more electronegative they are moving down the stairs and at the end they
move through most electronegative atom of all which of course oxygen.
The molecules are moving from molecule to another each time they move
they lose a little bit of energy . As electrons are being passed each of the
complexes is taking energy from the top and using that energy to drive
protons across the Inner Mitochondrial Membrane from Mitochondrial
Matrix to Inter-Membrane space. Building a gradient of protons, Proton
Gradient can work for the cell because protons want to come back in , they
want to move down concentration gradient . Only way protons can cross
Inner Mitochondrial Membrane is to go through a channel that is associated
with ATPases. Flow of protons down their concentration gradient drives
ATP Synthesis. Each of the Complexes are Redox molecules that are
capable of being reduced and oxidized. The Reason for that is they have a
lot of Organic cofactors like IRON,Sulfur , Copper . Comlex 1 has 46
subunits capable of taking electrons from NADH and pass it it down the
chain. So you generate a proton gradient by pumping protons across Inner
Mitochondrial Membrane, so inter-membrane space has more protons then
Mitochondrial Matrix, protons are pump from Matrix to Inter-membrane

Space generated a proton motive force . Which is driven by Membrane

Potential in other words a charge separation and also a ph gradient.
Protons want to come back through Inner Mitochondrial Membrane
because the gradient is driving them that way. And the only way they get
back through is through ATP Synthase
ATP SYNTHASE: Is a fascinating molecule that is able to couple the flow
of protons down their Concentration gradient with Synthesis of ATP from
ADP AND pi. Had different subunits it has an FO AND F1 UNITS.
FO Unit: Embedded in the Inner Mitochondrial Membrane, this is the part
that rotates around within the Inner Mitochondrial Membrane as protons
are coming thru it then there is a part that can undergo conformational
changes as it is being swivled.
F1 Unit: As protons come in it cause the F0 unit to role and keeo shifting,
while F0 unit is shifting it is making the F1 unit swilved. While F1 unit is
rotating it is changing conformation of subunits so they can bind ADP AND
PI and then bring them together to form ATP. Coupling flow of protons to
the movement of the stalk to conformational changes of F1 UNIT TO
SYNTHEZISE ATP.
Chemiosmosis: The use of energy in a H gradient to drive cellular work.
Proton gradient is generated by ETC in Mitochondria, chloroplasts, and
bacteria. Movement of protons down gradient drive rotation of subunits
withen F1 domain , which drives succession of conformational changes in
the alpha and beta subunit driving ATP Synthesis. So when protons in
cause the FO to rotate they affect the alpha and beta subunits of F1
drivinG ATP Synthesis.
F1 UNIT OF ATPASES: Has alpha beta and gamma subunits, the
alternating alpha and beta subunits create a Hydrophobic sleeve in the
middle of the Hexamer, and the gamma subunit fits there. The proton
gradient causes the gamma subunit to rotate inside the hexamer, and this
cause the Beta subunits to change their conformation. The beta subunits of
the F1 Unit go thru 3 conformational states: open, loose, and tight, which
define its affinity for adenine nucleotides. For ATP Synthesis, the loose site
must bind ADP AND PI, then a proton-driven rotation of the gamma subunit
causes a conformational change from loose to tight, which catalyzes ADP
and pi into ATP!!!!!.
24.Know the structure of the ER, the differences between smooth and
rough ER, and at least 5 ER functions.

Endoplasmic Reticulum: Is involved in processing and distribution of

proteins to various locations in the cell. The processing involves quality
control mechanisms. The ER is the source of secreted and membrane
proteins. The ER is the origin of most cellular membranes. The ER is the
site of lipid and sterol synthesis. The endoplasmic reticulum is the site of
Peroxisome membranes The ER is the site of calcium storage and
secretion. The ER has a calcium pump that pumps Ca against
concentration gradient into the Lumen of ER where it is stored.
Rough ER:Portion of the ER that is associated with proteins called Rough
ER what is rough of ER is the Ribosomes( Rough ER plays a role in
finishing of proteins that are going to be associated with Membranes or
secreted from the cell. Any membranous organelle like Nuclear Envelope
or ER itself, Golgi,Lysosome ,Plasma membrane any Membrane
associated or secreted protein is going to be finished in the ER ,folded
,modified . Proteins that are mutant or cant fold properly for whatever
reason, are held in the ER indefnitly or eventually just degraded. ER is
involved in maintaining protein quality control.
Smooth ER: Is not associated with proteins the smooth ER is involved in
Lipids. Sythesizing Lipids . Also breaking down Hydrophobic Compounds
or Toxic Compounds . Smooth ER plays a major role in detoxification. Liver
is the main detoxifying organ . Liver pulls off poisns that get into our
bloodstream from alcohol. Smooth ER in the Liver does this. The liver can
fail, if its Smooth ER has been overworked doing detoxification. Smooth
ER is also the place of Lipid Synthesis. HMG-COA Reductase involved in
synthesis of sterols , all of this take place in the Smooth ER as well.
Smooth ER ALSO RELEAES CALCIUM
Nuclear Envelope is also made in the ER both inner and outer Nuclear
Envelope it is rich in proteins that are part of nuclear pore complex and the
Lamins that anchor the chromatin to the Nuclear Envelope. There are also
portions of the ER that are involved in targeting vesicles and Lipids proteins
to other portions of the Endomembrane system.
25.Understand the role of signal sequences, SRP, SR, and the translocon.
Co-translational Transport into the ER: Some proteins are made on Free
Ribosomes in the Cytosol others are made on Ribosomes that become
associated with the Rough ER that means protein begins to be synthesized in
the Cytosol but then it is funneled into the ER and Rest of protein is made in
association with ER. Proteins that need to be translated in association with
Rough ER they have a specific sequence at the AMINO terminus of a protein

called the signal sequence it is the first 20 Amino acids and as that is coming
out of the Ribosome it is recognized as a signal sequence it is signaling that the
rest of the protein needs to be made in association with the Rough ER.
Remember there is only one pool of ribosomes; All of the Ribosomes are the
same. When a protein contains a signal sequence that signal sequence is
recognized by a signal recognition particle this recognizes the signal sequence
and interacts with it and when it does it pauses translation make the ribosome
stop reading the protin stop what you are doing I need to go over to the ER and
then you have to finish what you are doing . The signal recognition particle
binds and pause translation and then brings it over to the ER Membrane where
it interacts with (Signal Recognition Particle Receptor) SR when it does that the
whole complex becomes associated with a Huge Translocon and then the rest
of the protein is made in such a way that is being feed through this translocon
and accumulating in the ER, so the energy that the Ribosome is using to
synthesize the protein is powering through the translocon and inside the ER
there are Chaprones that are binding to the protein and pulling it along. The
Signal Recognition particle actually resembles one of the Elongation factors of
Translation. Instead of Elongation factors binding and translating the ribosome
the SRP doesnt translocate Ribosome it cause translation to pause , it
meantime it is getting the Ribosome with the Message and growing protein over
to the ER where it can funnel through the Translocon. From Slide: SRP binds
to the signal sequence and to the Ribosome, slowing protein synthesis. The
complex then binds to the SRP Receptor in the ER membrane . SRP is
released, passing ribosome to translocation channel. Channel inserts
polypeptide chain into membrane , starts transfer across lipid bilayer. Human
Signal Recognition Particle: 6 proteins and a 300nt RNA . Elongation arrests
by SRP binding to signal sequence happens because SRP structurally
resembles eEF2(which needs to bind for translocation to occur)
Signal Sequence: N-terminal 15-35 amino acids long. Stretch of at least 6
hydrophobic amino acids. Signal sequences are usually cleaved once they get
to ER. SRP and SR are examples of proteins that have this G-protein activity
they can exist in GTP or GDP BOUND STATE. When these proteins are in their
GTP bound state they can interact but then GTPASE hydrolysis put them in
GDP bound state and then they dissociate. Signal Recognition particle binds to
its Receptor but they are both G-Proteins.
SR is a Heterodimer, one domain binds Signal Recognition Particle other spans
ER Membrane.
Both SR and SRP have GTPase Domains.
So Protein being synthesized , signal sequences comes out the signal
recognition partible binds to it, that pauses translation, whole thing gets drag

over to the ER where signal recognition factor binds to receptor and then
become associated with Translocon and then the Ribosome and Translocon
are associated with each other in some way you dont need signal recognition
particle anymore that can diffuse away. The rest of the protein is made on
Ribosome that is sitting on top of Translocon, protein is being feed through the
translocon into the Lumen of the ER and BIPS is the name of the Chaporone
that is pulling it in. energy of protein synthesis and the BIP make sure protein is
moving in the rite direction.
Slide Co-translational tranlocation into the ER: Once nascent protein,
ribosome, srp are bound to srp receptor, srp release signal sequence to the
translocon in ER Membrane. SRP and SR then Hydrolyze their GTPS and
separate ,SRP recycles back to cytoplasm. The translocon is sec61 complex w
factors TRAM AND TRAP .Channel opens to the lumen,elongation of protein
synthesis pushes it through.BiP an ER luminal chaperone belonging to the Hsp
70 family binds protein. Binding an releaseing along with ATP hydrolysis allow
BiP to pull protein across membrane. BiP remains associated with unfolded
proteins to prevent misfolding and aggregation.
ER Sec 61 translocon : is oligomer of several Sec 61 complexes, which is a
heterotrimer of 3 transmembrane proteins only one Ribosome can bind . SEC
61 recognizes the signal sequence. So the protein is being made signal
sequence is held in Translocon and the rest of the protein is funneled through
and eventually a signal peptidase cleaves off the sequence and release mature
protein into the ER . So the mature protein has a new amino terminus.

26.Understand how ER membrane and ER lumen proteins are translocated

Different kind of proteins there are proteins that need to get into lumen of
ER there are also transmembrane proteins in ER. All transmembrane
domains are synthesized in the ER. All transmembrane proteins have to
situate themselves in the membrane while protein is being synthesized
because transmembrane domains are hydrophobic and the inside of the
ER is Hydrophillic. So transmembrane proteins is intergrated into ER
Membrane . N-terminal signal sequence initiates translocation . When
stop-transfer sequence enters channel, the channel discharges the
protein into the lipid bilayer. The stop transfer seq forms a stable
hydrophobic TM alpha helix, the N-terminal start transfer sequence is
cleaved by signal peptidase. So if a protein just has one Transmembrane
domain it is going to have this signal sequence that is going to get it to the
Translocn and the rest of the protein is going to feed in until stop sequence
says stop translocating thru the membrane so then Rest of protein is made

outside in cytosol. Many times proteins can have lots of transmembrane

domains. Trans- membrane protein is intergrated into the ER Membrane .
N-terminal signal sequence inititiates translocation. When stop transfer
sequence enter channel, the channel discharges the protein into the lipid
bilayer. The stop-transfer sequence forms a stabke hydrophobic TM alpha
helix. The N-terminal START Transfer sequence is cleaved by signal
peptidase. The amount of Transmembrane domains all has to do with
where and how many Start and stop sequences you have.
Soluble proteins destined for the ER Lumen are translocated fully across
the ER membrane in the Sec 61 translocon. Signal Peptidase, which is
associated with translocon, in ER lumen, cleaves signal sequence. The
signal peptide is degraded, the mature protein folds in the ER Lumen. Once
protein is in the lumen BiP and ER luminal chaperone belonging to the Hsp
7o family binds protein. Binding and releasing along with ATP hydrolysis
allow BiP to pull protein across membrane . BiP remains assocatied with
unfolded proteins to prevent misfolding and aggregation. A soluble protein
crosses the ER membrane and enters the lumen. The channel binds the
signal sequence, proteins crosses. Signal peptide remains bound to
channel, protein loops. Peptidase, onlumenal side of ER membrane,
cleaves , signal sequence is transferred to bilayer, where is ultimately
degraded. Once protein is released into the lumen channel closes. Transmembrane proteins can also use an INTERNAL START TRANSFER
SEQUENCE to integrate into ER membrane. Start transfer sequence is
recognized by SRP , which brings ribosome to translocon. When stoptransfer sequence arrives ,released into membrane both seqences start
and stop are part of mature protein. Proteins such as Ion Channels and
carriers rely on multiple start-stop sequences. Transmembrane domain
serves as an internal signal sequence, called a start-transfer sequence ,
recognized by SRP , brought to ER ,initiates translocaton, then protein
slides out of translocon. This internal start-transfer sequence is not
removed by signal peptidase, it remains part of the protein.
.
27.Understand the chain of events that enables glycosylation, disulfide
bond formation, and folding. Know the fates of properly folded and
misfolded proteins.
Proteins in the ER lumen have their signal sequence removed , they
undergo Glycosylation (addition of Oligosaccharides) . Oligosaccharyl
transferase adds core sugars to asparagine. They also undergo further
Glycosylation in the Golgi. A lot of N-linked Oligosaccharides. They also
undergo Disulfide bond formation by protein disuldide isomerase which

oxidizes SH of cysteine side chains to S-S. Keeps doing it until folding is

correct.1!!!! They also undergo Oligomerization in which Caperone BiP
binds Hydrophobic regions prevents aggregation, while proteins attempt to
fold properly. Proteins that are not folded properly or process properly or
are mutant are targeting for destruction. Adding carbohydrates to protein
prevent protein from aggregating on itself and folding improperly , by
glycolysated a protein, you add this large hydrophilic group to it and you
make it least likely for Hydrophobic patches to find one another and
aggregate. They also help proteins fold properly. Another thing DiSulfide
formation can only occur in the ER!!!!! These help stabilize the protein
by virtue of Covalent bond. Again Disulfide bond formation can only occur
in the ER!!!!!!!!! BECAUSE THEY WONT BE IMMEDIATELY REDUCED.
The ER SENSES how long a protein has attempted to fold itself in the ER
if it doesnt fold properly after a fix amount of times that protein is targeted
for export to cytoplasm and degradation in proteasome Proteasome is
site of protein degradation. The earliest steps in processing of a protein on
the co-translational pathway is addition of an oligosaccharide to the protein.
The oligosaccharide however is in the cytoplasm . A membrane bound
Lipid Dolichol phospahe binds to the oligosaccharide and flips it into the ER
lumen, it is then enzymatically attached to the protein. So we have
DOLICHOL Phosphate binds to oligosaccharide and flips it into the ER
LUMEN. And transferred to Asparagine the enzyme that does this is
Oligosaccharde Transferase. Many proteins need to be in the EndoMembrane system or ER to properly achieve their complicated 3D shape.
Proteins that help folding are members of these Calnexin family these all
help in ER protein folding these are all calcium binding proteins and they
all act like Chaprones. They help prevent the protein from aggregating. So
proteins that folded properly need to exported to the Golgo and Rest of the
CELL. If it is not folded properly, it needs a couple of trys if it cant fold
properly then it gets sent to the garbage dump. Misfolded proteins are
deglycosylated, ubiquitylated , and degraded by the proteasome. WAY
THE CELL knows that particular step a protein is in is that there are
enzymes that modify oligosaccardides either by taking glucose residues off
so that the protein can start folding and then adding glucose residues
back on if it needs to be refolded.
So once you add Oligosaccaride and the protein is unfolded , you now want
to get Chaporens , so first thing that happens to unfolded protein is that 2
glucoses come off this is what glucosidase does generating a
Monoglucosylated protein. is the signal to binding the protein Calnexin .
Calnexin can bind after two glucoses have been removed and when

Calnexin binds it help protein start folding. Calnexin is a chaperone it

facilitates protein folding and facilitates disuldife bond formation. So if
protein has folded properly other Enzyme that recognizes that Protein had
folded properly take off final glucose. So enzyme removes last glucose and
that tragets the protein for exit from the ER. But if enzyme looks at protein
and it doesnt look like it was properaly folded then that protein needs to go
back to the beginning and inorder to go back to the beginning it needs to
add glucose again so it can bind Calnexin again.
Inside the ER there are traces of Manosidase Enymes they remove
Mannose. ANY PROTEIN THAT IS PROPERLY FOLDED MOST LIKEY
WONT GET MANNOSE CLEAVED!!!!!!!!! When mannose is cleaved from
a protein that is a signal that the protein has been hanging out for too long
and that targets protein to Retrotranslocon that send bad protein out of ER
where it will be degraded. Again we have a cycle of Enzymes that remove
glucoses and add glucoses. Removing glucose allows the protein to be
recognized by the Calnexin, so that it can attempt to fold. If a protein is
folded properly it can exit the ER . If it is not folded properly it needs to add
glucose again so that Calnexin can recognize it again. When a protein
binds to Calnexin , that exposes protein to oxidoReductase that catalyzes
disuldie bond formation. IF ALL THAT GOES PROPERLY and protein is
folded properly remaining glucose are removed and protein is released . If
it is not folded properly it is recognized by this Glucosyl tranferase Enzyme
that adds more glucose so it can bind Calnexin and try a second time.
From Slide: After initial glycosylation, this cycle helps protein achieve
proper fold. 1st 2 glucoses come off, and monglucosylated product binds to
calnexin.
Calnexin binding exposes the protein to Oxidoreductase , which catalyzes
disulfide bond formation . When the remaining glucose is removed , protein
is releasesd . If it is not properly folded , it is recognized by UDP GT , which
glycosylates it so it can bind calnexin and try again.
28.Understand how an accumulation of unfolded proteins can trigger UPR,
and the outcomes of UPR activation (not the pathways, just the outcomes)
The ER SENSES stress . Stress is usually misfolded proteins. Misfolded
proteins are either re-folded or degraded. Mis folding can lead to ERAD
(ER assocoiated degradation or Unfolded Protein Response(UPR) Both
can occur simultaneously.
SO in UPD misfolded proteins in the ER lumen trigger the production of
chaperones and the expansion of the ER. So if you trigger Unfolded protein
response all of the BiP goes to work with all of these unfolded proteins and
then the sensors that trigger the unfolded response are no longer inhibited

by BiP . So in normal cells where ER is not swimming in Unfolded proteins

the BiP is keeping the unfolded protein response off. If some thing goes
wrong BiP leaves the sensors and goes an deals with the unfolded
proteins and the Sensors become active . This leads to an increase in
Chaporone Synthesis they lead to export of proteins from the ER, increase
in destruction of abnormal proteins and they slow down the rate of protein
synthesis All of these pathways are going to lead to production of more ER
, more Chaporone and better export of proteins out of the ER.
All are stress sensors that up regulate expression of genes involved in ER
function . UPR is inhibited by BiP , which associates with all 3 pathways ,
when BiP release sensors the pathways become active. Cells that can deal
with misfolded proteins often die. Some people believe that some diseases
have to with cells that cant do protein folding and trigger programmed cell
death or apotosis. So the ER had BiP and three additional systems for
detecting and ensuring quality control. Unfolded protein Response :
Misfolded proteins in the ER lumen trigger the production of Chaperones
and the expansion of the ER.
29.Understand the role of the ER in lipid synthesis, including the regulation
of cholesterol biosynthesis.
Most membrane lipids are synthesized in the ER except sphingomyelin and
glycolipids, and some unique lipids of mitochondria and chloroplasts .
Newly synthesized phospholipids are inserted into half of bilayer facing the
Cytosol , and then flipped into opposite leaflet by flippases. There are also
enzyme that modify lipids already present in the membrane . Newly
synthesized lipids are made on cytosolic face of ER. Some are flipped to
luminal side by translocators known as fippases and then transported to
Golgi,PM, etc in vesicles. The lipid compostion of different membranes vary
ER GOLGI PLASMA . All of these organelles vary in their lipid composition
but all are synthesized in the ER. There are several ways their lipids
compostion changes one is enzymes that modified the head of
phospholipids so it can change a phosphocholine to phosphoserine.
Enzymes can modify Lipids that already have been inserted in the
membranes. Also when vesicle can form it can form a Microdomain that
has a different lipid compostion. There are also these phospholipid transfer
proteins that can pick up lipids from one membrane and bring it to another.
Membranes can be alter by Lipid Carriers , can be altered by enzymes and
then can be altered by composition of a portion of a membrane that actualy
forms the vesicle. Cholesterol is a key component of plasma
membranes where does the cholesterol come from? It can be synthesize

inside the cell, or it can come from the food we eat and it is picked up by
receptor- mediated endocytosis. Cholesterol is bound to these LDL
receptors and internalized into the cell, so it can be made and it can be
absorbed from the food we eat. HMG-COA Reductase If there is not
enough cholesterol in your cells ? you want to turn on gene that encodes
this key enzyme. If there is too much cholesterol this enzyme is sent to the
Proteosome for destruction.
Bio-Synthesis of Cholesterol: Co-ordination of de-novo synthesis and
receptor meditated endocytosis. Begin in cytosol , HMG-COA reductase is
an intergral Membrane protein .
SREBP: Steroid regulator element binding protein
SCAP: SREBP cleavage activating protein
SCAP senses cholesterol, inhibits SREBP, and promotes HMG COA
reductase degradation
If there is not a lot of cholesterol in the Membrane that protein lets protein
that turns on HMG-COA reductase go and eventually gets to the nucleus
and turns on HMG-COA reductase. In the presence of high cholesterol this
protein that can drive HMG-COA REDUCTASE is trapped in ER because
the protein that senses cholesterol is trapping it. . If there is enough
cholesterol it will target HMG-CoA reductase for destruction by the
Proteosome .
So two mechanisms for High Cholesterol : One is destroying HMG-COA
Reductase and sending it to the proteasome. Other is blocking expression
of gene that encodes HMG-COA reductase.
In low cholestrerol , Cholesterol sensing protein isnt sensing any
cholesterol , so it is going to release SRE Binding protein which then gets
processed, it becomes activated as a Transcription factor and goes into the
Nucleus and turns on Expression of the enzymes that are required to
synthesize cholesterol also the enzymes that are involved of uptake of
cholesterol from food we eat,. So turning on Cholesterol Biosynthesis
Enzymes and up regulated Cholesterol receptors are two ways the body
copes with not having enough cholesterol in the ER Membrane.
FROM SLIDES : Hi Cholesterol in the ER Membrane is recognized by the
cholesterol sensing domain of SCAP along with Insig ,trap SREBP in the
ER membrane . This prevents SREBP from inducing the synthesis of
cholesterol biosynthesis enzymes. Also hi cholesterol targets HMG-COA
reductase for the ER Mediated proteolysis.
Low Cholesterol: Low cholesterol allows translocation to Golgi, and
cleavage of SREBP ,so that the TF portion can upregulate expression of
cholesterol Biosynthesis enzymes.

30.Understand how mitochondrial, peroxisomal, and ER defects can result

in human disease.
Inherited changes in mitochondrial DNA can cause problems with growth,
development, and function of the bodys systems. These mutations disrupt
the mitochondrias ability to efficiently generate energy of the cell. The
effects of these conditions are most pronounced in organs and tissues with
high-energy requirements (such as heart. Brain, and muscles) . Frequently
observed features include muscle weakness and wasting, problems with
movement, diabetes,kidney failure, heart disease.loss of intellectual
functions, hearing loss, and abnormalities involving the eyes and vision.
A buildup of non-inherited (somatic mutations) in mitochondrial DNA has
been associated with an increased risk of certain age-related disorders
such as heart disease,Alzhemer disease, and Parkinson disease.
Mitochondrial DNA accumulates mutations rapidly because oxidative
metabolism produces free radicals, which damage DNA. Additonally,
research suggest that the progressive accumulation of these mutations
over a persons lifetime may play a role in the normal aging process.
Mutations in four mitochondrial genes have been identified in people with
Leber hereditary optic neuropathy, these mitochondrial genes provide
instruction for making complex 1, necessary for oxidative phosphorylation,
so the mutations affect the generation of ATP within the mitochondria . It is
speculated that accumulations of mutations in mtDNA is a major cause of
aging. A premature-aging phenotype caused by increased mutations in
mtDNA The defective nuclear gene encodes the DNA polymerase
responsible for mtDNA replication. Diseases due to defeats in
Mitochondrial DNA happen in both inherited and Somatic( Results from
things that happen in your life) Parkinsons and Alzihmiers are due to
defeats in Mitochondrial DNA. All of Mitochondria are Maternally inherited.
Brain, heart and muscle require a lot of Mitochondria. Also Mitochondrail
genome doesnt have DNA damage repair surveillance mechanisms that
our chromosomal DNA has. It is not great at fixing mutations. In real life
during electron transport there are a lot of Reactive oxygen species lots of
molecules with unpaired electrons energetically active charged molecules
swimming around in Mitochondrial DNA. Mitochondrial DNA is an
environment that it susceptible to oxidation and damage. And it doesnt
have a robust repair mechanism. Complex 1 is they key complex that is
defective in Parkinsons disease. Mitochondrial DNA is replicated by a
particular DNA polymerase that is actually encoded in the Nucleus .
Example in which the a mutant mouse starts to age quickly because it has

a mutation in the Polymerase Enzyme that replicates mitochondrial DNA , it

is clear the defects in Mitochindria can contribute to premature aging.
Peroxisomal defeats that can contribute to Human disease:
An
essential Biosynthetic function of animal peroxisomes is to catalyze the first
reactions in the formation of plasmalogens which are the most abundant
class of phospholipids in myelin. Deficinecy of plasmalogens cause
profound abnormalities in the myelination of nerve cells, which is one
reason why many peroxisomal disorders lead to neurological disease. The
importance of peroxisomes is demonstrated by the inherited human
disease Zellweger syndrome , in which a defeact in importing protiens into
peroxisomes lead to severe peroxisomal deficiency. Mutations in at least
12 different genes, all involved in transport of peroxisomal proteins from
cytosol, have been characterized. Individuals with Zellweger syndrome
have cells that contain empty peroxisomes. Cannot break down toxic
substances in their brain, liver and kidneys and they die soon after birth.
X-linked adrenoleukodydstrophy is caused by lack of a single peroxisomal
membrane protein(ABCD1), which transports Very long chain fatty acids
into peroxisomes, where they are metabolized. In the absence of
peroxisomal import, the very lone chain fatty acids accumulate in the brain,
and destroy the myelin sheath of nerve cells , resulting in progressive
neurological dysfunction. In 2009 , successful gene therapy , using stem
cells from affected boys genetically modified w with ABCD1 gene. Some
gm cells went to brain, prevented further neuro-degeneration. `
ER DEFEATS: Many metabolic disorders, lysosomal storage diseases,
due to key enzymes unable to leave ER. Cystic fibrosis: CFTR cant get to
cell surface. Emphysema: alpha 1 antitrypsisn protects tissues from
elastase. Alpha 1 antitrypsin mutants cant fold and get out of the ER. For
example Lysosomes are organelles that contain digestive enzymes that
break down old macromolecules and organelles they are full of Hydrolytic
Enzymes and they recycle old and damage molecules. Lysosomal
enzymes are made in the ER and folded in the ER and packaged into
vescicles that goes to the Golgi and eventually to the Lysosome. Many
Lysosomal Enzymes like Tay Sachs in which there is a mutation in
Hydrolytic enzymes. Cystic Fibrous : Many mutations prevent Chloride Ion
transport from ever getting anywhere near the plasma membrane and it
cant even get out of ER properly because it cant fold properly Emphysema
: alpha 1 antitrysim protects tissues by inhibiting elastase, Antitrypsin cant
fold and get out of the ER.

Midterm 3 study guide:

This exam is not a cumulative final and only covers material since the last exam:
Chapters 21 Secretory Membranes, 23 Degradation, Ch24-27 Cell Signaling, Ch 40-44
Cell Cycle, and Cancer Biology

Know the functions of the secretory membranes.

Understand how lipid composition affects the thickness, permeability, and
transmembrane protein composition of phospholipid bilayers

Know the structure, composition, and functions of the Golgi apparatus.

Understand why constitutive and regulated degradation are so important.

Know the origin, composition, structure and functions of lysosomes.

Understand how proteosomes function, and the advantages of ubiquitin regulated
proteolysis

Explain why the proteosome is a valid target for cancer therapy.


Understand what ligand, receptor, signal activation, signal deactivation, transduction,
amplification, and response mean, and be able to give examples of each.

Understand how GPCRs and RTKs become activated and initiate signal transduction.
Know some examples of GPCRs and RTKs.

Understand the role of second messengers (Calcium, cAMP, IP3) in signaling.


Know the phases of the cell cycle (G1, S, G2, M) and the events that occur in each.

Understand what is meant by cell cycle regulation, and cell cycle checkpoints.

For each of the following you should be able to explain the signals that trigger the
checkpoint, and the signals that enable cells to pass checkpoints, and how they work.
G1 (R-point)
S (initiation of DNA replication)
G2 entry into mitosis
anaphase

Understand how genetics and biochemistry in model organisms led to the identification
of cell cycle regulators.


Know the landmarks of phases of mitosis (prophase, prometaphase, metaphase,
anaphase, telophase, cytokinesis)


Be able to give a basic definition of cancer

List the 6 hallmarks of cancer cells and explain why they contribute to the cancer
phenotype.

Know what oncogenes (Ras) and tumor suppressor genes (p53, Rb) are, know what they
do in normal cells, how they are altered in cancer cells, and why that contributes to
tumorigenesis.

Understand what is meant by the multistep model of tumorigenesis, and at least one
line of evidence that supports it.

Understand the initiator-promoter model of tumor progression, and how it explains
cancer incidence. Be able to distinguish between an initiator and a promoter.

Understand the role of chronic inflammation and obesity in cancer.

Understand how dietary choices might promote or prevent cancer.