BCH Lecture 8

7/27/2019 BCH Lecture 8

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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

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 forcells to carry out a signal)

Why make protein?

Lead to the development of the central dogma by Watson and Crick

The study of how DNA gets to proteins evolved into the field of molecular biology

Biochemistry: Lecture 8March-08-13

12:53 AM

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DNA is transcribed into RNA, RNA transcribed into protein

Process that converts RNA to DNA (reverse transcriptase)

Proteins seem to be the end point (nothing transcribing protein back into RNA)

Thought that process is irreversible and unidirectional

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

Promoters regions are typically "cis" acting elements - on the same strand of DNA being transcribed

Searches for initiation sites/promoter regions on a particular gene

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

Doesn't jump on and off like DNA polymerase does

Processive: one RNA molecule will carry out the entire transcription of the gene

Specific signals that will cause the RNA polymerase to stop transcription

Termination signals and prokaryotes and eukaryotes

Can be activators or repressors (can stop or activate transcription of a gene)

More for eukaryotic transcription, there are transcription factors that regulate the RNA polymerase function

Carry out the fundamental reaction of forming a phosphodiester bond

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Know the structures and numbering

On purines on 9, pyrimidines on 1

Base with glycosidic link

Have ribose (not deoxyribose)

Nucleotide with phosphates attached

Nucleoside structure:

Similar to DNA polymerase

RNA pol transcribes genes in the 5'->3' direction

Formation of phosphodiester bond

The release of PPi and hydrolysis to 2Pi drives the reaction forward

The incoming base is nucleophilically attacked by the hydroxyl oxygen on position 3' which attacks the alpha P

We don't need a primer for RNA polymerase because initiation site/promoter region that helps pol bind where it

starts transcription

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

Higher error rate (10

-5

) than DNA polymerase (10

-10

)

KNOW THESE NUMBERS FOR CALCULATION QUESTION (given the size of a DNA gene, how long with the RNA

polymerase take to transcribe it)

RNA speed is 40x less than DNA

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Deoxyribose missing 2'OH


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E.coli RNA polymerase - example of prokaryote

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

Large protein

Corresponding genes in E.coli are transcribed to generate protein (below)

Made of 4 different subunits - alpha, beta, beta prime and sigma 70

Core enzyme contains catalytic site: made of 2 alpha subunits, beta and beta prime

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

Holoenzyme includes the sigma subunit

Metal ions can be magnesium of manganate, but Mg is typically of choice

Manganate: any negatively charged Mg, typically referred to as MnO42-

Key AA residues that play a significant role in active site, key metal ions that help stabilize the proteins

Mg ion resides in the active site and coordinates with 3 aspartic residues

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

Incoming ribonucleotide beings in second magnesium to active site

Know the 3-letter codes for amino acids for exam (ex. Asp = aspartic acid)

Active site is similar to DNA polymerase

Technique determines specific regions on a strand of DNA that a DNA binding protein latches onto

Concept behind it:

How did they determine that RNA polymerase binds to different regions on DNA? - KNOW THIS SLIDE FOR EXAM

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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

Summary of 5 different promoter sites in 5 different genes

Similar initiation sites: tend to have the same nucleotides in particular positions

Transcription site is denoted +1

Genes are read 5'->3'

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

When we go to the left (up) from +1, we talk in negative numbers

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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

How does RNA polymerase recognize those core promoter regions?

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

Template strand also called antisense

Coding strand = sense

MAY SEE ON EXAM

RNA polymerase uses the template strand to make its transcript

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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Stronger the efficacy, the better the transcription

Efficacy denotes how capable that promoter is at carrying out a particular reaction

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 strong promoters have great efficacy

Gene may be transcribed one every 10 minutes in E.coli

Genes with weak promoters may have multiple substitutions in TATAAT

Distance for the optimal conservation of sequencesTypically the distance between the -10 to the -35 is 17-20 nt in distance

Promoters can be regulated through transcription factors

Enhances the stabilization of the protein on the DNA

Slightly increases the DNA binding affinity

For prokaryotes, there is an additional UP (upstream) element, which makes contact with the one of alpha subunits

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

The UP elements are seen in highly expressed genes (those that are constitutively active; being transcribed over

and over)

Note: sequences in red are conserved

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These are able to transcribe different genes at different rates

In addition to the standard promoter and UP element (on the left before sequence)

Don't need to memorize the sequence (won't ask 'What is the heat shock promoter sequence')

Genes such as heat shock proteins and involved in nitrogen metabolism have an alternative promoter

sequence at -35, -10

E.coli is unique because not only does it have the standard sigma 70 subunit, but has a wide variety of sigma

subunits

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

E.coli happens to be subjected to abrupt increases in temperature

Enhance transcription of genes that are involved in alternative sources of nitrogen metabolism

If you are nitrogen starved, can upregulate sigma 54

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

5 components, contains sigma subunit (makes contact between -35 and -10 element on dsRNA via transient )

3D representation of the RNA polymerase holoenzyme

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

Catalytic as it continually carries out reactions

After 8-10 oligonucleotides are synthesized, the sigma subunit comes off, finds another core RNA polymerase and

binds with it

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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)

This is called a transcription bubble

Unwinding at right end (at fork), and at the same rate rewinds at left end (at fork)

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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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'

Are synthesized 5'->3' because read a 3'->5' template

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

Elongation site is where ribonucleotides are added

RNA-DNA hybrid helix:

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 theRNA 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

Pre insertion site: ribonucleotides comes in and sits in this site

Conformational change

Inserted into insertion site

Study revealed that nucleotide insertion is a 3 step process:

This was discovered using streptolydigin, an antibody known for inhibiting bacterial RNA transcription initiation

processes

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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

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)

The assumption was that there was inactive pre-insertion site

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

No reaction is going to happen

Trigger loop still open

Ribonucleotide comes in and goes into the inactive preinsertion site

Key step: going from open trigger loop to closed trigger loop

Closing of the trigger loop (conformational change), pushes ribonucleotide from the preinsertion site into the

insertion site

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)

Intermediate step where the open trigger loop becomes a wedged trigger loop (helps in rachet-like motion),

and becomes open again

Incoming base is put into +1 site

The cycle repeats again

Nucleotide addition cycle:

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

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Open trigger loop

Closed trigger loop - triggers catalytic incorporation

Wedged trigger loop - provides translocation of the RNA-DNA hybrid helix

Know the different conformations:

Newly synthesized RNA strand (nacent RNA)

When you synthesize these regions, they start to base pair (self-complementary)

Formation of hairpin loop (stem with loop on top)

As it comes along, will synthesize a palindromic sequence (read in the same way in different directions CCGCC)

A-U base pairs are much weaker than the G-C base pairs; helps to destabilize the RNA-DNA hybrid helix

Termination signal is followed by many Us

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)

Termination signal in prokaryotes:

E.coli also has another mechanism to terminate gene transcription: protein dependent mechanism

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Sometimes need proteins to terminate signals

This is the case when it synthesizes rRNA (ribosomal RNA)

These proteins will help the RNA polymerase recognize a termination signal that it wouldn't be able to see on its

own

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)

Typically rRNAs have nomenclatures (10S, 13S. 17S)

When RNA pol transcribes rRNAs, in the absence of rho (p) (a particular protein identified in E.coli), will synthesize23S species of the rRNA

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

When included at the beginning of synthesis, RNA pol stopped transcribing at this point

Past first rho site, generates a larger fragment (13S rRNA)

Add rho in 30 seconds later

Another rho site, so rho is able to stop transcription here, generating 17S

Add in 2 minutes later

Study where they included the rho factor at different times with the RNA polymerase and DNA template

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

Hexameric ATP dependent helicase (bottom left)

What do rho proteins look like?

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

How does it help RNA pol cause termination?

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Antibiotics - inhibit bacterial transcription

Aug 6, 1881 - birth of Alexander Fleming

Saw zone of inhibition and extracted it -came up with penicillin

Sept 28, 1928 - working with staphylococci, went on vacation and saw petri dishes were filled with mold

Chain and Florey came along and took up his research - mass production of antibiotic

All won the Nobel prize in 1945

Green dot = active site of RNA polymerase in prokaryotes

Rifampicin binds to a pocket in the channel that houses the RNA-DNA hybrid helix

Good because doesn't affect eukaryotic polymerases

This pocket is highly conserved in bacterial RNA 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 - complex in nature; inhibits RNA transcription at the initiation site

Only inhibits RNA transcription initiation

Rifampicin sits in the pocket for the RNA-DNA hybrid helix

Has beta rings, inserts these rings (intercalates) into dsDNA

Makes dsDNA and ineffective template for RNA polymerase

At low quantities, effective at inhibiting transcription, but at high quantities has side effects because can

With this, it also affects DNA polymerase as well

Actinomycin: another antibiotic

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effect normally dividing cells

In the past, effective anticancer drug to stop rapidly dividing cells

Looking at RNA content in prokaryotes: cells have a large capacity to make rRNA, tRNA and mRNA

Why do we produce a lot of mRNA but keep only 3% of it in the cell?

Steady state levels show the rRNAs are highest and the mRNAs are the lowest

Other RNA molecules: ribonucleases

KNOW FOR EXAM

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

Typically the rRNA and tRNAs are synthesized together as a primary transcript

Hatch signs indicate the loop can be larger

rRNAs are drawn this way because they like to self base pair

Visual representation of primary transcript:

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Rnase III will cleave rRNAs from primary transcript (for 16S and 23S)

For 16S, it's M16

For 23S it's M23

For 5S it's M5

Smaller endonucleases that will trim the rRNAs

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

Ribose link is on position 1

Attached to ribose (not deoxyribose), so NOT T

Methyl group can be attached to U - ribothymidylate

Pseduouridylate - ribose moved from position 1 to position 5

Bases can undergo modifications - ex. Modification of U

Two methyl groups can be added to A in prokaryotes

Now moving from prokaryotic transcription to eukaryotic transcription

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mRNA doesn't go processing, cleavage or modification

Once synthesized, immediately undergoes translation

Prokaryotes:

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

Several promoter elements that are regulated through transcription factors

Can activate or repress transcription that is happening

Third factor - Promoter regions for eukaryotic genes are more complex

Eukaryote:

Different in terms of the location and the transcripts they synthesize

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 I responsible for synthesizing the majority of the rRNAs

snRNAs make up the splicosome

Strongly inhibited by amanitin (10 nanomolar - small quantity)

RNA pol II is responsible for the mRNAs and some of the snRNAs (small nuclear RNAs)

Inhibited by high concentration of amanitin (1 micromolar)

RNA pol III responsible for tRNAs and smaller 5S rRNA

3 eukaryotic RNA polymerases:

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Can have regulation of RNA pol through phosphorylation of the CTD region

Multiple repeats: serine and theronine residues like to be phosphorylated

KNOW TABLE FOR EXAM

RNA pol II from yeast

Multiple subunits, complex structure

Rpb2 subunit similar to beta

Rpb1 subunit (largest one) similar to beta prime

Rpb6 similar to sigma

Rpb3 is similar to alpha

Contains CTD

Analogous to prokaryotic RNA pol:

Fairly large

Don't have to know structure

Amanitin produced by mushrooms

Deadly poison, can inhibit RNA polymerase transcription at the elongation phase

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100 deaths/year - nanomolar concentrations to stop transcription of RNA pol II

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

Prevents closure of the active site

a-Amanatin does two things during elongation phase:

Preventing the open trigger loop from closing (no catalytic incorporation), prevents translocation step (going from

wedge to open trigger loop)

KNOW SITES WHERE IT ACTS IN ELONGATION CYCLE:

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

Summarizing key features of polymerases in cells

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:

Responsible for the rRNAs

TATA-like in nature; not a specific sequence but high concentrations of A and T

rInr - ribosomal initiator element

Constitutes the core promoter for RNA polymerase 1

UPE - upstream promoter element

Encompasses the transcription site (different from the -10 and -35)

Transcription start site (+1) is where the arrow starts

UPE regions tend to lie about -150 to -200 upstream of the ribosomal initiator element (large distance)

RNA pol I:

Two different sets of promoters: some have TATA box regions, some don't

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

TATA box is around -30 to -100 upstream of the initiator element (Inr) with +1 site = this is the core promoter

DPE found at about +30

May have enhancer regions to bind mediators

In TATA box less promoters, have initiator element and downstream promoter element (DPE) = core

promoter

RNA pol II:

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

Both or just one?

A block and B block = tRNA

RNA pol III:

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82% of the time there is a T, etc.

Small number indicate percent frequency of occurrence

Highly conserved promoter element for core promoter

Focus on RNAP II which have TATA box regions:

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

Genes that are highly transcribed (constitutive) also contain CAAT boxes, GC boxes

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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

Key figure to distinguish between prokaryotes and eukaryotes:

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

How does RNA polymerase start transcription in eukaryotes?

TBP recognizes the TATA box element, additional contacts made with the initiator element as well

First step is binding of TF II to DNA

Next, transcription factor A binds to TFD

Transcription factor B binds to massive complex

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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

RNA polymerase has unique CTD region which is rich in serines and threonines

BTA has a region that is UNphosphorylated

The assembly entire complex is called the basal transcription apparatus (BTA)

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)

Focus on TFIIA, B, D, TBP, E, H, F (don't have to worry about the rest)

TBP - key, starts assembly of BTATFIIH also phosphorylates the CTP region

Summarizes some the RNA TFs:

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Control occurs at the level of transcription

Ex. Hemoglobin

In addition, these allosteric proteins can also bind ligands - another area of regulation

Allosteric: bind to another site on the particular protein that can regulate it

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

A set of 3 genes transcribed under the control of a single promoter

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

Lac operon:

Operator region: allows for control of repressor protein, Lac i

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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 ofnegatively regulated genes - when repressor is bound, cannot transcribe lac operon

Repressor binds as a dimer to operator site

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

Our repressor is an allosteric protein

Betagalactosidase not only breaks down lactose into glucose and galactose, it carries out a minor side reaction by

generating allolactose

The inducer is allolactose

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

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

The lac operon has two operator regions

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 beta-

galactosidase, allolactose, permease, transacetylase

When you add the inducer in, the lac repressor comes off, and you get transcription of the lac operon

Lac repressor doesn't sit there all the time but falls off:

Can see formation of loop like structure

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

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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

Similar to the RNA polymerase sigma subunit, there is a key alpha helix that binds in the major groove of the DNA-

operator region

If lactose is present, it doesn't fit nicely into the major groove

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

When there is no lactose, the repressor is bound and it adopts the helix-loop-helix structure

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

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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

Adenylate cyclase takes ATP and makes cAMP

This occurs in addition to the repressor being bound to the operator

Once cAMP increases in the cell, will bind to CRP (cAMP regulatory protein; sometimes called CAP)

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

CRP helps RNA polymerase to bind to its promoter region

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

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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No glucose = CRP-cAMP bound

Lactose => produces allolactose => bind to repressor => falls off => transcription

1: don't have any glucose for E.coli, but have lactose

No CRP-cAMP because glucose present

Lac repressor is bound => no transcription

2: glucose present, no lactose

CRP-cAMP bound

Irrespective of whether we have CRP-cAMP bound, if the lac repressor is there, no transcription

Lac repressor bound => no transcription

3: no glucose, no lactose

No CRP-cAMP bound

No lac repressor => transcription

4: glucose, lactose

4 different conditions with differing glucose and lactose

Glucose can decrease the transcription rate almost by 50x

As you break down lactose, the glucose concentrations increase => slow down transcription

Condition 1 will have the higher transcription rate, because CRP-cAMP enhances the binding of RNA polymerase

*KNOW FOR EXAM

Prokaryotes are simple - have repressors and activator

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Simplified view of a promoter region on eukaryotic gene

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

RNA polymerase doesn't just bind to gene of interest, but has to be recruited through transcription factor II factors

Eukaryotes have a wide variety of TF that can regulate gene transcription

Can be up to 1kb away

Bridge activator to RNA polymerase through co-activators

Wide variety of proteins associated with co-activators

Can bind activators; larger proteins that increase the efficiency of transcription

Silencer regions: repressors that bind and decrease the efficiency of RNA polymerase transcription

Enhancer regions:

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

Summary of TBP binding:

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TTAACA; same on corresponding strands

Two fold access of symmetry = regions in red

One of the operator regions on the lac operons

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

What the regions look like when TFs bind:

Unique protein motifs with DNA binding proteins

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

Common: Helix-loop-helix

Tandem repeats of small sets of alpha helices that are coordinated through zinc binding

Zinc ion coordinates to cysteine residues in the proteins

See alpha helix, loop and zinc

One of the regions is an alpha helix

Enters major groove, interactions with DNA binding region

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Leucine zippers are two long alpha-helical like structures

DNA binding region contains basic residues (arginine R, glycine K)

Leucine zipper - leucine present at every 6th residue; can see in red on structure

Amino acid sequence:

Hydrophobic, so can diffuse across the cell membrane

Binds to soluble nuclear receptors - involved in regulating gene expression

Structure of estradiol

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Bottom right: purple helix = helix 12

Ligand binding region made of many helices

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

Structure of a 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

Estradiol will bind to ligand binding pocket in receptor

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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

Does ligand binding effect its binding to DNA? It doesn't

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 co-

activators

Open up the gene to increase transcription for RNA polymerase

These are part of a larger family of proteins called P160, catalyze a series of reactions that lead to chromatin

remodelling

Why do we even need estradiol if it doesn't change DNA binding affinity?

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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

This type of mechanism can be used to make drugs that can stop the transcription of genes

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BCH Lecture 8