BIOL3401 Lecture 8 Eukaryotic Transcription

How many RNA polymerase(s) do a Eukaryotic nuclei contain? and how are they separated?

3 RNA polymerases and separated by ion-exchange

What kind of ion exhange is DEAE Sephadex?

Anion Exxhange

List out the types of of eukaryotic polmerases, what it transcribes and it’s location,

RNA polymerase 1 transcribes large rRNAs in the nucleolus

RNA polymerase 2 transcribes pre mRNAs in nucleoplasm

RNA polymerase 3 transcribes tRNA, small 5s RNA and small nuclear RNAs in nucleoplasm

How many groups and hence subunits in Polymerase II?

3 Groups and 12 subunits

Name the groups in the Polymerase II.

1. Core

2. Common

3. Nonessential subunits

With the names of the 3 groups in the Polymerase II. List the subunits that is in each group and also list the ones that isn’t in one.

1. Core (Rpb1, Rpb2, Rpb3)

2. Common (Rpb5, Rpb6, Rpb8, Rpb10, Rpb12)

3. Nonessential subunits (Rpb4, Rpb9)

The Rpb7 and Rpb11 do no fall into any groups and although it is required for polymerase activity, if it is mutated, then the mutants are not viable.

What does Core Polymerase II responsible for?

The structure and function of bacterial core subunits such as Beta', Beta, Alpha

Which group is found in all RNA polymerases?

Common

What group is conditionally dispensable for enzymatic activity?

Nonessential subunits

There are two different forms of RNA polymerase II: IIO and IIA. What are their subunits, difference and function?

IIA: Rpb1’ CTD unphosphorylated and initially binds to the promoter

IIO: Rbp1’ CTD phosphorylated and carries out elongation

Wgat is the function of the Clamp in a RNA polymerase II?

In a free polymerase without DNA binding, the clamp opens to allow access to the active site.

In a elongated complex with DNA-RNA hybrid, the clamp is closed to ensure the enzyme is processive so that it is able to transcribe a whole gene without falling off the terminating transcription prematurely.

Wgat is the straight state?

The active site opened for the addition of a nucleotide

Wgat is the bent state?

After addition and coincident with translocation the helix bridge shifts to bent

When does the trigger loop come into play?

The trigger loop only comes into play when the correct substrates occupies site A and makes several important contacts with substrates.

What is the purpose of the trigger loop?

To stabilize the substrate’s association with the active site and contribute to the specificity of the enzyme.

What -TP conformations does the trigger loops have?

GTP, ATP, UTP and no nucleotide

Wgat is the difference between the 3 eukaryotic RNAPs?

-Different structures

-Transcribing different classes of genes

How many classes of promoters are there?

3 classes

How many parts does the class 2 promoter have?

2 parts

Core promoter

Proximal promoter

What do those promoters do?

Core promoter attracts general transcription factors and RNAP 2 at basal level and sets the transcription start site and direction

Proximal promoter helps attract general transcription factors and RNAP and includes promoter upstream elements at transcription start site

Wgat are the core promoter elements

TATA BOX

INR

DPE

DCE

MTE

TFIIB

Wgat does the TATA box do?

Involved in positioning of transcription start

The distancne between TATA box and transcription start remains same

Wgat are silencers and enhancers

Cis-acting DNA elements not part of promoter but very influential in transcription (tissue specific)

How does silencers work?

causes chromatin to coil up into a condensed inactive form and so stopping transcription of neighbouring genes

Wgat do transcription factors aid in?

Transcription factors aid in binding Eukaryotic RNA polymerases to promoters

Wgat classess of TF are there

general and specific (activators)

Wgat do RNAP Class 2 factors do?

Combine RNAP to form preinitation complex (PIC)

wgat are the 6 general TF?

TFIIA

B

D

E

H

F

Wgat do these TFII do?

TFIID, with the help of TFIIA, binds to the TATA box creating a DA complex

Then TFIIB binds to the complex

After that TFIIF helps the RNAP bind to the a region extending from -17bp to +17bp at least.

TFIIE and TFIIH binds forms DABpolFEH preinitiation complex.

Wgat does TFIID contain?

-TATA box binding protein (TBP)

-Several TBP associated factors specific for class 3 (TAFIIs)

Wgat curve is the TATA box bent?

80 Degree

Wgat does the opening of the minor groove do?

It aids in local DNA melting that is part of forming an open promoter complex

Why is TBP important

1. Assembly of a preinititaion complex

2. TBP plays an organizing role in preinitiation complexes on MOST types of eukaryotic promoters

3. Specificity of TBP is governed by different TAFs with which it associates when binding to various promoters.

How does TFIIB bind to TBP?

by C terminal to TATA box and N terminal to RNAP2.

Wgat is the last general transcription factor joining the complex?

TFIIH

Wgat does the TFIIH do?

1, Phosphorylates C terminal CTD of RNAP2. (Essential for initation → elongation)

2. Unwinds DNA at the transcription start site to create the transcription bubble. (Helicase activity)

Wgat different forms of RNAP2 are there?

IIA (Rpb1’ CTD unP)

IIO (Rpb1’ CTD P)

Elongation complex continues elongation RNA when:

Polymerase CTD is further P’d by TEFb and NTPs are continuously available.

Is ATP needed in Elongation?

Yes

During transcriptional activation process, what P’s multiple different sites on CTD of RNAP2.

several protein kinases and TFIIH

Why is multiple sites on CTD P’d?

So the P’d CTD tail changes binding int between RNAP2 and other general transcription factors.Hence, enabling breaking away from most TF bound to promoter

Wgat does TFIIS do?

Stimulates Elongation

Stimulates Proofreading of Transcript

Wgat happens when RNAP2 pauses at a pause site?

RNAP backtracks to left causing transcription arrest that RNAP cannot recover from

Latent Ribonuclease act of Poly. stimuated by TFIIS cleaves off extruded 3’ end of Nascent RNA (PROOFREADING)

Resume transcription

How does termination occur in the Torpedo model?

RNA is transcribed downstrea from polyA tail site and attacked by 5-3 RNase. When RNase catches up to Poly, it triggers dissociation from DNA template

How does termination occur in the Allostericmodel?

Is highly processive.

Then when PolyA signal passed → less processive.

Alternation due to modification or conformational change.

What are the two well-conserved elements in Class 1 promoters? and is spacing important?

-Core element surrounding transcription start site

-Upstream promoter element (UPE) 100bp upstream

-Yes

How does transcription VIA RNAP1 happen?

C1 promoters recognized by two TF, core binding factor (SL1 in humans) and UPE binding factor(Helps bind core promoter). Poly1 terminates and relies on endonuclease to cleave nascent RNA.

How many types of C3 promoters?

3 types

What is so special about C3 promoter types?

T1 and T2 have promoter that lie within genes.

T3 promoter resembles those of C2 promoters.

How does chromatin accessibility and transcription work?

When chromatin is permissive, it can initiate seq-specific remodelling and establishes an open chromatin confromation.

How are chromatin structure allterantion directed by activators?

1. By binding to DNA sites bound to upstream of promoter that is inacessible by chromatin and recruting histone acetylase and adding acetyl grps to residues within histone tails.

2. Recruting nucleosome remodeler that alters strcuture of nucelosomes  around promter making it inacessible.

This create packing of nucleosome creates binding sites for proteins to carry bromodoamins (Appropriate recognition domains), Allowing binding of T machinary to Promoter.

Wgat is FACT?

heterodimer of Spt16and SSRP1 by dealing with histones in its path and is able to dismantle nucleosomes ahead of transcribing RNA and reassemble them behind.

Wgat does FACT speciafically remove?

H2A-H2B dimer

Wgat does SPT6 do?

Binds to Histone H3 and belived to aid in nucleosome assmebly.