Biochemistry: Lecture 8

March-08-13 12:53 AM

Know the nucleic acid structures for final exam Final exam: 3 hours, 35%, all MC

Primary transcript = mRNA = extensively modified in eukaryotes, but not in prokaryotes Although the processes tend to be similar, there are major differences between the two

Genetic information is encoded in DNA, blueprint for proteins Why make protein? Secondary and tertiary structures of proteins allow them to carry out specialized functions and keep the cell viable Proteins have a variety of functions: constitute structural integrity (make up the cytoskeleton, actin and myosin), transport proteins (hemoglobin transport oxygen), enzymes (catalyze reactions), signal transduction (receptors for cells to carry out a signal) The study of how DNA gets to proteins evolved into the field of molecular biology Lead to the development of the central dogma by Watson and Crick DNA is transcribed into RNA, RNA transcribed into protein
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 DNA is transcribed into RNA, RNA transcribed into protein Thought that process is irreversible and unidirectional Process that converts RNA to DNA (reverse transcriptase) Proteins seem to be the end point (nothing transcribing protein back into RNA)

Share an evolutionary ancestry Eukaryotic RNA polymerase has more subunits (7-12), prokaryotes have less (5) Carry out the same processes of initiation, elongation and termination

Searches for initiation sites/promoter regions on a particular gene Promoters regions are typically "cis" acting elements - on the same strand of DNA being transcribed Unwinds dsDNA because it needs to make a ssDNA template for the RNA polymerase to transcribe Inserts the correct ribonucleotides Process is unidirectional: once RNA polymerase latches onto the DNA, will go from 5'->3' to transcribe that particular gene Processive: one RNA molecule will carry out the entire transcription of the gene Doesn't jump on and off like DNA polymerase does Termination signals and prokaryotes and eukaryotes Specific signals that will cause the RNA polymerase to stop transcription More for eukaryotic transcription, there are transcription factors that regulate the RNA polymerase function Can be activators or repressors (can stop or activate transcription of a gene) Carry out the fundamental reaction of forming a phosphodiester bond

Know the structures and numbering

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Know the structures and numbering

Deoxyribose missing 2'OH

Nucleoside structure: Base with glycosidic link On purines on 9, pyrimidines on 1 Have ribose (not deoxyribose) Nucleotide with phosphates attached

RNA pol transcribes genes in the 5'->3' direction Similar to DNA polymerase The incoming base is nucleophilically attacked by the hydroxyl oxygen on position 3' which attacks the alpha P Formation of phosphodiester bond The release of PPi and hydrolysis to 2Pi drives the reaction forward We don't need a primer for RNA polymerase because initiation site/promoter region that helps pol bind where it starts transcription Higher error rate (10-5) than DNA polymerase (10-10) This is tolerated because many copies of the genes are translated - if there is an error in 1 or 2 of the proteins being made, it won't have as detrimental of an effect as an error in DNA RNA speed is 40x less than DNA KNOW THESE NUMBERS FOR CALCULATION QUESTION (given the size of a DNA gene, how long with the RNA polymerase take to transcribe it)

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 E.coli RNA polymerase - example of prokaryote Large protein Da: standard unit for indicating mass on an atomic or molecular scale; one-twelfth of the mass of an unbound neutral atom of carbon 12 1.6 x 10-27 kg Made of 4 different subunits - alpha, beta, beta prime and sigma 70 Corresponding genes in E.coli are transcribed to generate protein (below) Core enzyme contains catalytic site: made of 2 alpha subunits, beta and beta prime Holoenzyme includes the sigma subunit Sigma subunit is counterproductive, as it decreases the DNA binding affinity If you lose the sigma subunit, the core enzyme binds with a higher affinity Present to find promoter regions; after 10 nucleotides have been synthesized, it is released

Active site is similar to DNA polymerase Key AA residues that play a significant role in active site, key metal ions that help stabilize the proteins Metal ions can be magnesium of manganate, but Mg is typically of choice Manganate: any negatively charged Mg, typically referred to as MnO42 Mg ion resides in the active site and coordinates with 3 aspartic residues Incoming ribonucleotide beings in second magnesium to active site Will coordinate with the aspartic residues and the other Mg to stabilize incoming ribonucleotide When formation of phosphodiester bond occurs, the PPi leaves the active site and takes the Mg ion with it Know the 3-letter codes for amino acids for exam (ex. Asp = aspartic acid)

How did they determine that RNA polymerase binds to different regions on DNA? - KNOW THIS SLIDE FOR EXAM Technique determines specific regions on a strand of DNA that a DNA binding protein latches onto Concept behind it: Take dsDNA
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 Take dsDNA One of strands radiolabel with P32, incubate two strands with Dnase (do it long enough so you can nick the strands one nucleotide at a time) Add DNase again to get another cleavage to generate a smaller fragment When run on an agarose gel, can separate radiolabelled strands using an audioradiogram and detect the different sizes of the bands Repeat under the same conditions, but add in specific DNA binding protein (ex. RNA polymerase) We know that is binds to a specific promoter region Take the DNase, nick the DNA - generate larger to smaller products When it comes to the point where the RNA polymerase is bound, it hides the DNA and cannot generate fragments in this area If run on the gel, see bands that are missing - in this region is where the RNA polymerase is binding the ds DNA Can do this for any DNA binding protein For exam question: if given a gene sequence that is radiolabelled, fragments of a gel and told you that DNase was nicking it one nucleotide at a time, should figure out where on the sequence RNA polymerase is bound to

Similar initiation sites: tend to have the same nucleotides in particular positions Summary of 5 different promoter sites in 5 different genes Transcription site is denoted +1 Genes are read 5'->3' When we go to the left (up) from +1, we talk in negative numbers At -10 (10 nucleotides upstream of transcription site), we have a conserved region When you take an average of these sequence, we tend to see TATAAT in the -10 region 82% of the time you see the T in the position, etc. This average of consensus sequence is derived by David Prinbrow; called Prinbrow box Constitutes the core promoter In addition, at -35 another consensus sequence Together the -35 and -10 constitute the core promoter for RNA polymerase in prokaryotes

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How does RNA polymerase recognize those core promoter regions? There is a helix in the sigma subunit that tends to dissociates after 10 nucleotides are synthesized Helix has a role in -10 recognition in addition to -35 Key tyrosines (Tyr), tryptophan (Trp), glutamine (Glu), threronine (Thr) and arginine (Arg) residues that make transient hydrogen bonds with the bases located in the -10 region Another region in the sigma subunit that does the same for the -35

A gene is typically read 5'->3' Complementary is 3'->5' When RNA polymerase opens/unwinds the double stranded DNA, end up with coding strand and template strand RNA polymerase uses the template strand to make its transcript Template strand also called antisense Coding strand = sense MAY SEE ON EXAM Used the 3'->5' template strand to make a 5'->3' transcript The mRNA is the same in sequence as the coding strand, except have a U instead of the T

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Efficacy denotes how capable that promoter is at carrying out a particular reaction Stronger the efficacy, the better the transcription Genes with strong promoters have great efficacy Whether they are efficient or not is another story If have a strong promoter, have a high correspondence to TATAAT, and transcribe genes frequently Genes with weak promoters may have multiple substitutions in TATAAT Gene may be transcribed one every 10 minutes in E.coli Typically the distance between the -10 to the -35 is 17-20 nt in distance Distance for the optimal conservation of sequences Promoters can be regulated through transcription factors For prokaryotes, there is an additional UP (upstream) element, which makes contact with the one of alpha subunits Enhances the stabilization of the protein on the DNA Slightly increases the DNA binding affinity

Note: sequences in red are conserved

The UP elements are seen in highly expressed genes (those that are constitutively active; being transcribed over and over) Increases transcription by increasing DNA binding with RNA polymerase About 40-60 nt upstream A and T rich, n denotes other nucleotides - not highly conserved but does provide additional binding for the alpha subunit

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 E.coli is unique because not only does it have the standard sigma 70 subunit, but has a wide variety of sigma subunits These are able to transcribe different genes at different rates In addition to the standard promoter and UP element (on the left before sequence) Genes such as heat shock proteins and involved in nitrogen metabolism have an alternative promoter sequence at -35, -10 Don't need to memorize the sequence (won't ask 'What is the heat shock promoter sequence') E.coli happens to be subjected to abrupt increases in temperature It increases the production of sigma 32, which recognizes alternative promoter sequences for the heat shock genes Heat shock genes are chaperones and help proteins fold - if you heat up E.coli, can help fold back some denatured proteins If you are nitrogen starved, can upregulate sigma 54 Enhance transcription of genes that are involved in alternative sources of nitrogen metabolism Some E.coli have 8-15 different types of sigma subunits - depending on the type of situation the prokaryote might find itself it, it can upregulate different sigma subunits

3D representation of the RNA polymerase holoenzyme 5 components, contains sigma subunit (makes contact between -35 and -10 element on dsRNA via transient )

Searches for promoter regions very rapidly: latches on, starts to look for promoter sites Random walk: will latch on and keep going until it finds it (processive) One RNA polymerase will completely transcribe that particular gene (will not get on and off the DNA strand) Because of this its rate of binding is high After 8-10 oligonucleotides are synthesized, the sigma subunit comes off, finds another core RNA polymerase and binds with it Catalytic as it continually carries out reactions

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 Holoenzyme migrates along DNA template strand looking for promoter, find it, latches on 2nd picture: closed promoter complex - reversible at this stage (can come off and no transcription can happen) 3rd picture: formation of the open promoter - unwinds the dsDNA, transcription is irreversible (committed to transcribing that particular gene) 4th picture: initiation of RNA synthesis, most typically starts with a purine (A or G)

Holoenzyme - has sigma subunit Core enzyme - doesn't In terms of ligand binding, a lower Kd = higher affinity Thus core enzyme has higher affinity, so holoenzyme will have a larger Kd (approx 105 times higher)

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 Start bringing in ribonucleotides Once 8-10 are synthesized, the sigma subunit falls off and finds another core RNA polymerase to continue transcription The RNA polymerase moves down the template strand, carries out the rest of the transcription of the gene

Yellow portion = RNA polymerase RNA polymerase unwinds about 17 base pairs when it's making this open promoter complex (approx 1.6 turns of dsDNA) Unwinding at right end (at fork), and at the same rate rewinds at left end (at fork) This is called a transcription bubble

Left: it's easier to break A-T bonds, so will typically see an A-T rich region that is unwound

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Only 5' end has gamma labelled P

Are synthesized 5'->3' because read a 3'->5' template Know this to be true because studies have been done where radiolabelled ribonucleotides (P32 on gamma phosphate), in the gene that is transcribed it is only detected at 5'

Initiation - starts with an A or G for prokaryotes Incoming ribonucleotides 3'OH nucleophilic attack at the alpha phosphate Formation of phosphodiesterbond Release of PPi, hydrolyzed to 2Pi (drives reaction forward) Next nucleotide can enter at bottom Repeats over and over again until gets to the end of the gene

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 RNA polymerase in yellow, unwinding occurs at the same rate as DNA rewinding RNA-DNA hybrid helix: Elongation site is where ribonucleotides are added RNA-DNA helix turns as it is being unwound/rewound RNA-DNA hybrid helix passes into a channel in the RNA polymerase At the end of channel, have separation of template strand from newly synthesized nascent strand RNA-DNA helix is 8bps, about half the size of the replication bubble (17bp)

Separation of the template strand from the nascent RNA is carried by the helix-loop-helix that is conserved in the RNA pol Green and red: channel; helps to separate the two as they exit the polymerase

Elucidated using RNA pol II (from eukaryotes), can also apply to the RNA pol found in prokaryotes Within the RNA polymerase there are 2 key structures - bridge helix (not main focus) and trigger loop Trigger loop is an alpha helix loop helix structure, add nucleotides to the RNA polymerase gene Key conformational changes that occur in the active site that helps with the insertion of the ribonucleotides Study revealed that nucleotide insertion is a 3 step process: Pre insertion site: ribonucleotides comes in and sits in this site Conformational change Inserted into insertion site This was discovered using streptolydigin, an antibody known for inhibiting bacterial RNA transcription initiation processes Antibody binds into the insertion site
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 Antibody binds into the insertion site Using x-ray crystallography and in vivo assays, they found that nucleotides were still bound in the active site The assumption was that there was inactive pre-insertion site Resides in the insertion site, but you can still see the ribonucleotides bound Close to the active site, but not close enough to carry out the fundamental reaction (formation of the phophodiester linkage) Different conformations of the bridge helix and trigger loop suggest their involvement in facilitating translocation (moving on for the next cycle to begin)

Nucleotide addition cycle: At the top = DNA template in blue +1 = initiation site RNA product with 3 nucleotides added to it Not focusing much on bridge helix, but trigger loop (alpha helix loop helix structure) Trigger loop is in open position Ribonucleotide comes in and goes into the inactive preinsertion site No reaction is going to happen Trigger loop still open Closing of the trigger loop (conformational change), pushes ribonucleotide from the preinsertion site into the insertion site Key step: going from open trigger loop to closed trigger loop In insertion site, catalytic incorporation Nucleophilic attack, PPi is released Have to move everything over, to free up +1 site for the next incoming ribonucleotide Translocation process that occurs (circled in box) In pre-translocation stage, the closed trigger loop opens up again (undergoes conformational a change) Bridge helix and open trigger loop play a role in pushing the nascent RNA strand over by one nucleotide (rachet type mechanism) Incoming base is put into +1 site Intermediate step where the open trigger loop becomes a wedged trigger loop (helps in rachet-like motion), and becomes open again The cycle repeats again Summary: ribonucleotide comes into the pre-insertion site, closing of trigger loop, catalytic incorporation, Ppi release, pre-translocation, opening of trigger loop, wedge conformation with ratchet like mechanism, pushing everything one nucleotide away, opening up again for an incoming ribonucleotide Know the different conformations:
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Know the different conformations: Open trigger loop Closed trigger loop - triggers catalytic incorporation Wedged trigger loop - provides translocation of the RNA-DNA hybrid helix

Termination signal in prokaryotes: Newly synthesized RNA strand (nacent RNA) As it comes along, will synthesize a palindromic sequence (read in the same way in different directions CCGCC) When you synthesize these regions, they start to base pair (self-complementary) Formation of hairpin loop (stem with loop on top) Termination signal is followed by many Us A-U base pairs are much weaker than the G-C base pairs; helps to destabilize the RNA-DNA hybrid helix Once the polymerase senses the hairpin loop structure, it stalls and loosens its grip on the DNA and terminates transcribing of that particular gene Hairpin loops and U is plenty to signal the RNA polymerase to stop transcribing The signal for termination lies within the nascent RNA strand (not coded for in the DNA gene)

E.coli also has another mechanism to terminate gene transcription: protein dependent mechanism Sometimes need proteins to terminate signals
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 Sometimes need proteins to terminate signals These proteins will help the RNA polymerase recognize a termination signal that it wouldn't be able to see on its own This is the case when it synthesizes rRNA (ribosomal RNA) Typically rRNAs have nomenclatures (10S, 13S. 17S) The number dictates the size; the larger the number the larger the size of the rRNA "S" = sedimentation coefficient (large the number, larger the weight of the rRNA) When RNA pol transcribes rRNAs, in the absence of rho (p) (a particular protein identified in E.coli), will synthesize 23S species of the rRNA Study where they included the rho factor at different times with the RNA polymerase and DNA template When included at the beginning of synthesis, RNA pol stopped transcribing at this point Only generated the 10S species (smaller than 23S) Must have been a rho site, because the rho factor helps stops transcription of that particular rRNA Add rho in 30 seconds later Past first rho site, generates a larger fragment (13S rRNA) Add in 2 minutes later Another rho site, so rho is able to stop transcription here, generating 17S Can generate 4 different types of rRNAs using a protein dependent termination On it's own, the RNA polymerase does not see the rho site, but in combination with the rho factor it can generate different rRNA species Protein independent termiation, protein dependent termination In eukaryotes transcription, there are no identifiable rho factors - rho factors are isolated from E.coli

What do rho proteins look like? Hexameric ATP dependent helicase (bottom left) How does it help RNA pol cause termination? Searches for a C rich site on nascent RNA Latches on, looks for C rich site (sometimes called a rut site - rho utilization site) Once it finds C rich region, ATP hydrolysis occurs, moves quickly along the nascent RNA chain and catches up to the RNA polymerase Once it finds RNA-DNA helix, it uses its helicase activity to dissociate RNA from DNA It doesn't bind to dsDNA, it binds to ssRNA The signal resides in the nascent RNA produced, and not the DNA template

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Antibiotics - inhibit bacterial transcription Aug 6, 1881 - birth of Alexander Fleming Sept 28, 1928 - working with staphylococci, went on vacation and saw petri dishes were filled with mold Saw zone of inhibition and extracted it -came up with penicillin Chain and Florey came along and took up his research - mass production of antibiotic All won the Nobel prize in 1945 Rifampicin - complex in nature; inhibits RNA transcription at the initiation site Green dot = active site of RNA polymerase in prokaryotes Rifampicin binds to a pocket in the channel that houses the RNA-DNA hybrid helix This pocket is highly conserved in bacterial RNA polymerases Good because doesn't affect eukaryotic polymerases Prior to initiation, if rifampicin is able to get into channel, can stop bacterial RNA polymerase transcription Can't stop transcription if the RNA-DNA hybrid helix is already fit into the channel Only good at stopping transcription initiation - if elongation has taken place (more than 3-4 nucleotides have been synthesized), this isn't effective at all Pocket is about 12 angstroms away from active site (small distance)

Rifampicin sits in the pocket for the RNA-DNA hybrid helix Only inhibits RNA transcription initiation Actinomycin: another antibiotic Has beta rings, inserts these rings (intercalates) into dsDNA Makes dsDNA and ineffective template for RNA polymerase With this, it also affects DNA polymerase as well At low quantities, effective at inhibiting transcription, but at high quantities has side effects because can effect normally dividing cells
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effect normally dividing cells In the past, effective anticancer drug to stop rapidly dividing cells

KNOW FOR EXAM Looking at RNA content in prokaryotes: cells have a large capacity to make rRNA, tRNA and mRNA Steady state levels show the rRNAs are highest and the mRNAs are the lowest Why do we produce a lot of mRNA but keep only 3% of it in the cell? Other RNA molecules: ribonucleases

Typically the rRNA and tRNAs are synthesized together as a primary transcript All on the same region of the gene using a single promoter Transcript will include several rRNAs and one or two tRNAs The yellow regions are spacer regions - transcripts that will be excised These are arranged in tandem repeats (after this is another promoter, 16S, tRNA, 23S, 5S etc.) These undergo cleaving, processing, base modification However, the mRNAs are unmodified and cleaved unprocessed

Visual representation of primary transcript: rRNAs are drawn this way because they like to self base pair Hatch signs indicate the loop can be larger Rnase III will cleave rRNAs from primary transcript (for 16S and 23S)
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 Rnase III will cleave rRNAs from primary transcript (for 16S and 23S) Smaller endonucleases that will trim the rRNAs For 16S, it's M16 For 23S it's M23 For 5S it's M5 tRNAs are excised from primary transcript using Rnase P for 5', Rnase D will cleave 3' end Another enzyme that will add CAA onto the 3' - this isn't coded for from the primary transcript This shows the processing of the tRNAs and rRNAs

Bases can undergo modifications - ex. Modification of U Ribose link is on position 1 Methyl group can be attached to U - ribothymidylate Attached to ribose (not deoxyribose), so NOT T Pseduouridylate - ribose moved from position 1 to position 5

Two methyl groups can be added to A in prokaryotes

Now moving from prokaryotic transcription to eukaryotic transcription

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Prokaryotes: mRNA doesn't go processing, cleavage or modification Once synthesized, immediately undergoes translation Eukaryote: This doesn't occur in eukaryotic cells - more complex type of regulation in terms of gene transcription First factor - Has to do with nuclear membrane Genes are transcribed mRNA is processed within the nucleus: 5 methyl G cap, poly A tail, splicing Transported out for translation on the ribosomes The membrane allows for spatial and temporal regulation of transcript - dictates when gene is transcribed and how it's taken out of the cell Second factor - Because of these modifications, it takes a while to undergo translation process Third factor - Promoter regions for eukaryotic genes are more complex Several promoter elements that are regulated through transcription factors Can activate or repress transcription that is happening

3 eukaryotic RNA polymerases: Different in terms of the location and the transcripts they synthesize RNA pol I responsible for synthesizing the majority of the rRNAs Affects of a poison (amanitin) that can inhibit some of the polymerases, or not inhibit one of them RNA pol I is insensitive to this poisonous compound RNA pol II is responsible for the mRNAs and some of the snRNAs (small nuclear RNAs) snRNAs make up the splicosome Strongly inhibited by amanitin (10 nanomolar - small quantity) RNA pol III responsible for tRNAs and smaller 5S rRNA Inhibited by high concentration of amanitin (1 micromolar)

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 Multiple repeats: serine and theronine residues like to be phosphorylated Can have regulation of RNA pol through phosphorylation of the CTD region KNOW TABLE FOR EXAM

RNA pol II from yeast Multiple subunits, complex structure Analogous to prokaryotic RNA pol: Rpb2 subunit similar to beta Rpb1 subunit (largest one) similar to beta prime Rpb6 similar to sigma Rpb3 is similar to alpha Contains CTD

Fairly large Don't have to know structure

Amanitin produced by mushrooms Deadly poison, can inhibit RNA polymerase transcription at the elongation phase 100 deaths/year - nanomolar concentrations to stop transcription of RNA pol II
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 100 deaths/year - nanomolar concentrations to stop transcription of RNA pol II

a-Amanatin does two things during elongation phase: Prevents closure of the active site Prevent incorporation of nucleotide because the open trigger loop cannot close, and the ribonucleotide cannot be delivered into the insertion site, no more nucleotide incorporation Stops translocation step - stops wedge trigger loop from opening again, no more nucleotide incorporation KNOW SITES WHERE IT ACTS IN ELONGATION CYCLE: Preventing the open trigger loop from closing (no catalytic incorporation), prevents translocation step (going from wedge to open trigger loop)

Summarizing key features of polymerases in cells U1,U2,U4 and U5 snRNAs make up the splicosome A large percentage of activity of the polymerases is devoted to the synthesis of the rRNAs Don't need to know mitochondrial/chloroplast

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The prokaryotic promoter is simple: -10 and -35 regions, sometimes a UP element The eukaryotic promoters are different for the 3 RNA polymerases, and different from prokaryotes Some of the common elements in the eukaryotic promoter regions: RNA pol I: Responsible for the rRNAs rInr - ribosomal initiator element TATA-like in nature; not a specific sequence but high concentrations of A and T UPE - upstream promoter element Constitutes the core promoter for RNA polymerase 1 Transcription start site (+1) is where the arrow starts Encompasses the transcription site (different from the -10 and -35) UPE regions tend to lie about -150 to -200 upstream of the ribosomal initiator element (large distance) RNA pol II: Two different sets of promoters: some have TATA box regions, some don't TATA box is around -30 to -100 upstream of the initiator element (Inr) with +1 site = this is the core promoter Enhancer regions that lie far upstream (can be 1 kilo base pair away from the initiator element; very large distance) Enhancer regions bind mediators that can either activate or repress transcription In TATA box less promoters, have initiator element and downstream promoter element (DPE) = core promoter DPE found at about +30 May have enhancer regions to bind mediators RNA pol III: Responsible for tRNA and 5S Specific consensus sequences (A block and C block) A block and C block lie downstream of initiator elements = 5S RNA A block and B block = tRNA Both or just one?

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Focus on RNAP II which have TATA box regions: Small number indicate percent frequency of occurrence 82% of the time there is a T, etc. Highly conserved promoter element for core promoter

Genes that are highly transcribed (constitutive) also contain CAAT boxes, GC boxes Variable in terms of distance, reside between -40 to -150 in promoter region Genes that are highly expressed generally contain a CAAT and GC box as additional promoter elements for RNA polymerase

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How does RNA polymerase start transcription in eukaryotes? Key figure to distinguish between prokaryotes and eukaryotes: In prokaryotes, RNA pol binds to the DNA itself (and searches for promoter regions) RNA polymerase in eukaryotes must be recruited to DNA by transcription factors There are key transcription factors (TF II, for RNA polymerase II) In transcription in eukaryotes, there is a large complex protein called TFIID - approx 70kd in size (large protein) Within that large complex, smaller protein (30 kd) called the TATA box binding protein (TBP) TBP will recognize the TATA box promoter element for the RNA pol II transcription initiation process

First step is binding of TF II to DNA TBP recognizes the TATA box element, additional contacts made with the initiator element as well Next, transcription factor A binds to TFD Transcription factor B binds to massive complex Next is transcription factor F
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Next is transcription factor F F recruits RNA polymerase Once bound, RNA pol will recruit E and H (in that order) Order: D, A, B, F, RNA pol, E, H The assembly entire complex is called the basal transcription apparatus (BTA) RNA polymerase has unique CTD region which is rich in serines and threonines BTA has a region that is UNphosphorylated

CTD is phosphorylated by TFH Stabilizes the BTA and recruits processing enzymes needed for the mRNA (splicing factors, poly A polymerase for poly A tail, enzyme needed for 5 methyl G cap) Once phosphorylation, initiation begins and RNA polymerase can start transcribing DNA is open once phosphorylated, transcription factors dissociate (except F)

Summarizes some the RNA TFs: Focus on TFIIA, B, D, TBP, E, H, F (don't have to worry about the rest) TBP - key, starts assembly of BTA TFIIH also phosphorylates the CTP region

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 Control occurs at the level of transcription

Allosteric: bind to another site on the particular protein that can regulate it Ex. Hemoglobin In addition, these allosteric proteins can also bind ligands - another area of regulation 4 types of situations in terms of gene expression control:

Ligand causes binding of the activator, helps to facilitate RNA polymerase binding = transcription of gene Absence of activator and ligand = no transcription

Ligand can have a negative effect Binds activator causing it's release, causing RNA polymerase to stop transcribing that gene

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Free repressor from promoter, allowing RNA polymerase to transcribe gene

Ligand can have a stimulatory effect and bind repressor

Operon: A set of 3 genes transcribed under the control of a single promoter Lac operon: Single promoter and 3 genes transcribed Lac Z: produces protein beta-galactosidase; helps in the catabolism of lactose (because down into monosaccharides) Lac Y: galactosidase permease; helps bring in lactose into the cell of E.coli (prokaryotic operon?) Lac A: transacetylase protein; acetylates any unused lactose allowing it to be eliminated Operator region: allows for control of repressor protein, Lac i Transcription of this gene occurs further upstream, and Lac i has its own promoter
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 Transcription of this gene occurs further upstream, and Lac i has its own promoter When Lac i is transcribed, it forms the repressor that binds to the operator region, preventing transcription of the lac operon Lac operon is a set of negatively regulated genes - when repressor is bound, cannot transcribe lac operon

Repressor binds as a dimer to operator site

Our repressor is an allosteric protein If a ligand is present (an INDUCER in this situation), it can bind to the repressor, allowing it to come off and for the genes to be transcribed

The inducer is allolactose Betagalactosidase not only breaks down lactose into glucose and galactose, it carries out a minor side reaction by generating allolactose

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Two types of mechanisms that can regulate this type of transcription - one involving lactose, the other involving glucose

The lac operon has two operator regions Operator regions are close together A dimer will bind one of the operator regions, another dimer will bind the second one Repressor binds as a tetramer Forms a loop like structure: RNA polymerase cannot transcribe this Lac repressor doesn't sit there all the time but falls off: Quickly re-binds to operator region But because it falls off, the RNA polymerase is able to transcribe the lac operon at least once = basal levels of betagalactosidase, allolactose, permease, transacetylase When you add the inducer in, the lac repressor comes off, and you get transcription of the lac operon

Synthetic DNA - put in the sequence for one of the operators at the beginning of the DNA, and another about 500 bp away, add in repressor Can see formation of loop like structure

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Similar to the RNA polymerase sigma subunit, there is a key alpha helix that binds in the major groove of the DNA operator region Key arginine residue that will base pair with the C-G in the major groove for the operator gene Structure is a helix-loop-helix that fits into the major groove of the lac repressor Because it binds as a dimer, there are two of them

If lactose is present, it doesn't fit nicely into the major groove When there is no lactose, the repressor is bound and it adopts the helix-loop-helix structure Without repressor, you don't see the helix-loop-helix conformation very well; once it is able to bind into major groove it adapts this structure and forms the hydrogen bonds with the arginine and G-C base pairs

If we look at the amount of beta-galactosidase produced when we add lactose into a medium containing E.coli, we can see that the amount of protein produced is directly proportional to cell growth Once we add lactose into medium, cells start to grow (glucose used for energy)
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 Once we add lactose into medium, cells start to grow (glucose used for energy) When lactose is removed, no more B-galactosidase is being produced

When the cells don't have any glucose (no food), EIII (enzyme 3) in E.coli will take the phosphate from phosphoenolpyruvate and use it to phosphorylate adenylate cyclase Adenylate cyclase takes ATP and makes cAMP Once cAMP increases in the cell, will bind to CRP (cAMP regulatory protein; sometimes called CAP) This occurs in addition to the repressor being bound to the operator CRP helps RNA polymerase to bind to its promoter region There is a CRP-cAMP binding region for these genes in addition to the lac operon Think of as a UP element - further upstream from the promoter, helps the RNA polymerase bind to promoter

Helps bind the RNA polymerase, so there is a greater interaction and an increase in the efficiency of transcription of that gene

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4 different conditions with differing glucose and lactose 1: don't have any glucose for E.coli, but have lactose No glucose = CRP-cAMP bound Lactose => produces allolactose => bind to repressor => falls off => transcription 2: glucose present, no lactose No CRP-cAMP because glucose present Lac repressor is bound => no transcription 3: no glucose, no lactose CRP-cAMP bound Lac repressor bound => no transcription Irrespective of whether we have CRP-cAMP bound, if the lac repressor is there, no transcription 4: glucose, lactose No CRP-cAMP bound No lac repressor => transcription Condition 1 will have the higher transcription rate, because CRP-cAMP enhances the binding of RNA polymerase Glucose can decrease the transcription rate almost by 50x As you break down lactose, the glucose concentrations increase => slow down transcription *KNOW FOR EXAM

Prokaryotes are simple - have repressors and activator

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Eukaryotes have a wide variety of TF that can regulate gene transcription Simplified view of a promoter region on eukaryotic gene RNA polymerase doesn't just bind to gene of interest, but has to be recruited through transcription factor II factors TATA binding protein = part of TFIID Basal transcription apparatus bind to core promoter with TATA box regions Coding region on bottom: eukaryotic gene that needs to be transcribed Enhancer regions: Can be up to 1kb away Can bind activators; larger proteins that increase the efficiency of transcription Bridge activator to RNA polymerase through co-activators Wide variety of proteins associated with co-activators Silencer regions: repressors that bind and decrease the efficiency of RNA polymerase transcription

Summary of TBP binding: Binding in TATA box region; -30 to -100 nucleotides from initiator (+1) element Subscripts show frequency of conserved region Mutations in the TATA box markedly decreases the efficiency of RNA transcription Part of TFIID; very large complex

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What the regions look like when TFs bind: One of the operator regions on the lac operons Two fold access of symmetry = regions in red TTAACA; same on corresponding strands Arginine can bind a G-C base pair, glutamine can interact with A-T in terms of the lac repressor binding the operator regions Common feature of TFs: binds as dimers because of two fold axis of symmetry

Unique protein motifs with DNA binding proteins Common: Helix-loop-helix Comes into the major groove of DNA One of the helices is called the recognition helix, this is the one that makes the interaction with the DNA in the major groove

Tandem repeats of small sets of alpha helices that are coordinated through zinc binding Zinc ion coordinates to cysteine residues in the proteins One of the regions is an alpha helix See alpha helix, loop and zinc Enters major groove, interactions with DNA binding region

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 Leucine zippers are two long alpha-helical like structures Amino acid sequence: DNA binding region contains basic residues (arginine R, glycine K) Leucine zipper - leucine present at every 6th residue; can see in red on structure

Structure of estradiol Hydrophobic, so can diffuse across the cell membrane Binds to soluble nuclear receptors - involved in regulating gene expression

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Structure of a receptor: Ligand binding region made of many helices Bottom right: purple helix = helix 12 In terms of protein structure, in there receptor there is a region for transcription activation (bind activators and facilitate transcription) DNA binding region consists of two binding motifs (two zinc fingers; zinc coordinated with 4 cysteine residues) Hormone binding: binds estradiol

Estradiol will bind to ligand binding pocket in receptor In the absence of ligand, helix 12 protrudes from the receptor at the bottom When the ligand binds, causes a conformation change and pushes the helix into a groove on the side of the receptor

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Does ligand binding effect its binding to DNA? It doesn't Irrespective of whether estradiol is present or not, the DNA binding affinity of the receptor for the DNA doesn't change DNA binding region can bind in the absence of estradiol; separate domains

Why do we even need estradiol if it doesn't change DNA binding affinity? Estradiol indirectly affects gene expression Cellular zippers - 2 of them, binding as dimers Can see protruding helix 12; affinity doesn't change much whether estradiol is there or not; receptor is still bound to the DNA for the particular gene we want to transcribe When the estradiol binds and causes a conformational change in helix 12, it facilitates the recruitment of coactivators These are part of a larger family of proteins called P160, catalyze a series of reactions that lead to chromatin remodelling Open up the gene to increase transcription for RNA polymerase

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This type of mechanism can be used to make drugs that can stop the transcription of genes Ex. Tamoxifen Used in estrogen-mediated breast cancers Resembles estradiol - benzene rings, but has an extra piece on bottom Fits into the ligand binding site, but extra portion protrudes Cannot get helix-12 to fold into the pocket on the side of the receptor, causes distortion, cannot get recruitment of co-activator and thus no chromatin remodelling Can think of estradiol as an agonist for this receptor, and tamoxifen as an antagonist

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