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“Transformation
principle”
Convincing evidence!
X-ray diffraction analysis
Precursor during NA biosynthesis
+cell bioenegetics
(a) Linkage of two nucleotides by the
formation of a 5' phosphodiester bond,
producing a dinucleotide. (b) Shorthand
notation for a polynucleotide chain
A weak electrostatic interaction between
a covelently bonded H atom and an atom wth an
ushared electron pair
TWO RULES
*A+G=C+T
A detailed
view
1 Angstrom
(Ā)=0.1 nm)
Sedimentation Equilibrium
Centrifugation (Density
Gradient Centrifugation):
Neutral buoyant density;
no further migration
To fractionate the gradient, successive samples are eluted from the bottom of
the tube. Each is measured for absorbance of UV, producing a profile of the
sample in graphic form.
(GC bp.s are more compact and dense)
Increase in UV absorption vs temperature
(the hyperchromic shift) for two DNA
molecules with different GC contents (melting
profile)
Lesser GC
content
Tm: Melting
temp.
Greater GC
content
k: second-order
rate constant
Cot1/2: Half reaction time; when one half of the DNA is present as ds fragments
(directly proportional to genome size; useful to assess genome size of organisms)
Cot Analysis III
Molecular hybridization between
DNA fragments and RNA
↓
Localize to chromosomes with microscopy. Or after hyb. with biotinylated
probe, fluorescein coupled to avidin is reacted with cytological specimen
FISH
Electrophoretic separation
Semiconservative replication of DNA
The Meselson–Stahl experiment (1958)
Always proceeds from the end with 5’-P to the 3’-OH end (5’-P of incoming nucleotide is
attached to 3’-OH of the previously added nucleotide)
Helical unwinding of DNA during replication as
accomplished by DnaA, DnaB, and DnaC
proteins (helicases)
The lagging template strand is looped in order to invert the physical (not biochemical!)
direction of synthesis
Summary of DNA synthesis at a
single replication fork
(stabilize ss
DNA)
(11 bases)
Six identified* :
(At least 13 different )
*also ζ pol in repair, **low processivity; adds only a short DNA sequence to the primer,
then polymerase switching, ***main polymerase
Each eukaryotic chromosome
contains many replicons
• 250-400 in yeast, 25.000 in mammals. Origins in
yeast: Autonomously Replicating Sequences (ARSs)
• Hard to measure size of average replicon since
adjacent ones fuse. Thought to be from 40 to 200
kb.
• No termination signals. Terminate by meeting one
in opposite direction.
• Active replicons are clustered; 20-80 adjacent
replicons are bound to a group of sp. proteins:
Origin Recognition Complex (ORC). 300-500 active
at any given time in mammals.
• If all replicons active, replication could be complete
in 1 hr, but S phase lasts about 6 hr. This implies
that only about 15% are active at any given time.
In eukaryotes, Pol α is required for the
initiation of replication at origins and for
the priming of Okazaki fragments during
the discontinuous synthesis of the lagging
strand. Pol α exists in a stable complex
with DNA primase; indeed, they copurify
during isolation. The primase synthesizes
the RNA primers, which are then
extended with deoxyribonucleotides
by Pol α to produce an RNA–DNA chain
about 30 nucleotides in total length.
These RNA–DNA primer chains are then
extended by Pol δ . Pol completes
the replication of the lagging strand, while
polymerase ε catalyzes the replication of
the leading
strand.
NOTICE!!
• Smaller replicons (100-200 kb) in
eukaryotes.
• Okazaki fragments: 1000-2000 nts in E.
coli, 100-150 nts in eukaryotes.
• Rate of synthesis: 100 kb/min in E. coli,
20X faster than that in eukaryotes; still it
takes much shorter time to replicate whole
genomic DNA in eukaryotes.
The telomere problem: The difficulty
encountered during the replication of the
ends of linear chromosomes
Telomerase
Telomerase:
– is a ribonucleoprotein
– Its single RNA molecule (159 base RNA) provides an AACCCC (in
mammals) template to guide the insertion of TTGGGG
– Its protein component — called hTERT in humans ("human TElomere
Reverse Transcriptase") — provides the catalytic action
– Thus telomerase is a reverse transcriptase; synthesizing DNA from an
RNA template
Aging :
– In most eukaryotic somatic cells, the telomerase activity stops
shortly after the cell differentiates.
– After this, the chromosomes gradually shorten with each
division (telomere length: cellular clock)→cell senescence→cell
death
– Malignant cells maintain telomerase activity: Aging vs
immortality (cancer)
The telomere solution
unorthodox
H bonding
DNA-dependent RNA Polymerase
(rpo A, B, C, D genes)
The interaction of RNA polymerase with
the promoter: Consensus sequences
Promoters typically consist of a 40 bp region on the 5'-side of the transcription start site
(-10 region)
Eukaryote
• Promoter positions differ for each polymerase- not all upstream
• Main consensus sequence TATA box (-25) and CAAT box (-60 to -120) Plants
have AGGA instead of CAAT
• RNA POL I – rRNA
• RNA POL II – mRNA
• RNA POL III –5S rRNA, tRNA
• Enhancers to increase transcription
• In eukaryotes, the population of primary transcripts in a nucleus is called
heterogeneous nuclear RNA (hnRNA) because of the large variation
in the sizes of the RNA molecules present. Initial product of transcription
is not usable; primary transcript to be processed. Longer life time
(hours/days)
An interpretive
drawing of an electron
micrograph of a hybrid
molecule: Heteroduplex
Chicken ovalbumin gene
with 7 DNA introns and
mature ovalbumin mRNA
Post-transcriptional RNA processing in
eukaryotes (from hn RNA/RNP in
nucleus→mature RNA)
Poly(A)
polymerase
10 – 30 nts 20 – 40 nts
Endonuclease
AAUAAA
ATP
Polyadenylate polymerase
RNA+ nATP = RNA-(AMP)n + nPPi
PPi
AAUAAA AAA(A)n
80 – 250 A’s
Polyadenylate polymerase
1. The 3’ OH of a free
guanosine attacks the
phosphate at the 5’
splice site resulting in
the 1st trans-
esterification
Splicing mechanism:
– Involves lariat formation similar to group II
– Requires RNA-protein complexes (snRNPs)
= small nuclear ribonucleoproteins (“snurps”)
Spliceosome
Structure
• 60S dynamic structure (may contain ~ 50 proteins)
• snRNPs
• splicing factors
• assembly of spliceosome requires ATP
Function
• Provide high accuracy of splicing
Splicing defects
• Estimation: 15% of all genetic diseases associated
with mutated splice sites
snRNAs
Length
snRNA Function
(nts)
Binds 5’ splice site, then 3’ splice
U1 165
site
Binds the branch site and forms
U2 185
part of the catalytic center
U4 116 Masks the catalytic activity of U6
AG
U1 U2
U5
U4
U6
Another group of Introns: tRNA
and/or archaeal introns
found in the nuclear tRNA of eukaryotes and
in archaeal tRNA, rRNA and mRNA.
Splice variants:
HOMEWORK!
(summaries; each max. 1 page)
RNA Editing (late 1980s):
Pre-mRNA seq. is changed prior to
translation
1. Substitution Editing
•Also C to U
2. Insertion/Deletion
editing
•Pre-edited RNA base pairs
with a guide RNA on both
sides of the region to be
edited.
•In mt RNA of protozoa (e.g.
Leishmania) and slime molds
(e.g. Physarum)
•The guide RNA is also
transcribed from mt genome;
provides a template for the
insertion of uridines.
•The mRNA produced by the
insertions is complementary to
the guide RNA.
C to U editosome:
Protein complexes
UNIVERSAL,
but...
Codon Usage Bias (Codon Preference)
Differences in frequency of occurrence of synonymous codons in coding DNA.
A balance between genomic GC content, mutational biases and natural selection for
translational optimization (rate & accuracy)
The Codon Adaptation Index (CAI)is the most widespread technique for
analyzing Codon usage bias by measuring the deviation of a given protein coding
gene sequence with respect to a reference set of genes. Ideally, the reference
set in CAI is composed of highly expressed genes, so that CAI provides an
indication of gene expression level under the assumption that there is
translational selection to optimize gene sequences according to their expression
levels. The rationale for this: highly expressed genes need to compete for
resources (i.e. ribosomes)in fast-growing organisms leading to highly expressed
genes using mostly codons for tRNA species that are abundant in the cell.
Overlapping genes: In some
viruses and mtDNA
Loops contain modified bases
(can not form bp)
3’ACC
Unusual bases (Rare bases;
odd bases)
Purine hypoxanthine
(Zig-zag plane)
The higher-level assciation of β sheets has been implicated in
(Right-handed formation of the protein aggregates and fibrils observed in
many human diseases, notably the amyloidoses such as
helix; spiral conformation)
Alzheimer's disease.
(3-D); twists, turns, loops
around itself
Co-linearity
-covalent disufide bonds
-polar, hydrophilic R
groups interact with water
-nonpolar, hydrophobic
R-groups inside the molecule
interact with each other
(Oligomeric protein)
multi-subunit;
e.g. polymerases
Protein tertiary structure
- folding and packing of secondary structure elements, super-
secondary structure elements, domains
- tertiary folding is stabilized by some non-covalent interactions
- H-bonding
- ionic interactions (salt bridges)
- van der Waals forces
- ‘hydrophobic’ interactions
- can be stabilized by covalent bonds: disulphide linkages
Unstable in cytosol as a
-Cys reducing env., but are
-Cys -S mostly found in extracellular,
secreted and periplasmic
-Cys proteins as well as
Oxidizing -Cys -S for cytosolic proteins under
agent oxidative stress!!!
CystEine Cystine
We are able to predict the secondary structures of proteins from their sequences quite
well (75% accuracy), but fail miserably at predicting the tertiary structures.
4 subunits (each is a protomer;
oligomeric protein
Most proteins demonstrate a mixture of
these structures
• Fibrous proteins: Elongated, strand-like (filamentous), insoluble
in water, weak acids and weak bases but soluble in strong acids
and alkalis. A single unit or structure is repeated multiple times.
The peptide chains are bound together by strong intermolecular
hydrogen bonds. Highly resistant to digestion by enzymes.
inteins as small
as 134 amino acids
can splice out
of precursor proteins
(monofunctional inteins)
Inteins as Mobile Genetic Elements: Homing
Endonuclease Activity
• 50-300 aa
• functional capability
• unique folding
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
One or more
exons is copied
(duplication;
but part of
genes) and
inserted
elsewhere to
give rise novel
arrangements
of exons (no
need to
reinvent
wheel!)
11-146
Two proteins that are similar
in certain regions
Tissue plasminogen activator (PLAT)
Coagulation factor XII (F12).
Kringle domains are believed to play a role in binding mediators (e.g., membranes, other proteins or
phospholipids), and in the regulation of proteolytic activity.
Exon Shuffling
• Transposons can drive "exon-shuffling" to
create new genes
Proteins are the molecular tools
for most cellular functions
• Protein folding
• Proteolytic cleavage
• Chemical modification
• Intein splicing
Post-translational processing and
modification-FOR FUNCTION
Protein processing
Cleavage
• N-terminus methionine entirely or its formyl group is usually removed in prokaryotes.
In eukaryotes, methionine entirely can be removed.
• Preproinsulin to insulin (occurs in cells)
• Pre-prothrombin to prothrombin (in cell) cleaves off leader or signal peptide
• Prothrombin to thrombin (occurs in blood)
Modification
• N-terminus methionine’s amino group is removed or acetylated
• Disulfide bridge (CySH+CySH to CyS-SCy)
• Glycosylation (covalently; e.g. antigens in ABO blood system)
• Methylation or acetylation
• Lipid-linking
• γ-carboxylation
• Phosphorylation (of –OH groups of certain aa.s by kinases ) of e.g. tyrosines, for
ionic bonding with other molecules
• Hydroxyproline (in collagen)
• Complexation with metals (e.g. Hb)
Protein modifications:
requirements for activity
- cleavage and covalent modifications of proteins (often after synthesis) but
may also be co-translational
I. CLEAVAGE
some proteins require sections of the polypeptide chain
to be removed for correct maturation.
peptide sequences
disulfide
- di-sulphide bonds hold the two peptides H2N- bonds
together H2N-
Covalent Modifications
Sometimes proteins are covalently modified after synthesis
These modifications can be:
1. Required to obtain the active conformation (e.g.. collagen)
2. Used to control the activity of a protein
(e.g. histones, signal transducing proteins, etc.)
O
O
C OH
C R
HN
OH
R N
H
OH
Prothrombin, histones
Prothrombin: Glutamate gamma-Carboxy Glutamate
This requires Vitamin K; No vitamin K No Blood Clotting
O
O
H2N CH
CH C OH
H2N CH
CH C OH
CH
CH 2
CH
CH 2
CH 2
CH
CH
CH
C O
O C C O
O-
O- O-
O C O C
H2 H2 H2 H2 H2 H2 H2 H2 H
CH C C C C NH
NH 2
2 CH C C C C N CH
C
3
NH 2 NH 2
O
Post translational modification
in rough ER and golgi
Post translational modifications in golgi and rough ER
include glycosylation:
e.g. DnaK
e.g. DnaJ-stimulates
ATP hydrolysis by DnaK
Nascent
chain-associated complex
Trigger
factor
Prefoldin
Hsp 60 in mitochondria/GroES-
GroEL complex in bacteria