eukaryotic rna polymerases

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Gene Expression Eukaryotic Gene Transcription 9/18/08 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics [email protected]

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Gene Expression Eukaryotic Gene Transcription 9/18/08 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics [email protected]. Eukaryotic RNA Polymerases. Three DNA dependent RNA polymerases: RNA Pol I, II, and III All 3 are big, multimeric proteins (500-700 kD) - PowerPoint PPT Presentation

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Page 1: Eukaryotic RNA Polymerases

Gene Expression

Eukaryotic Gene Transcription9/18/08

Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics

[email protected]

Page 2: Eukaryotic RNA Polymerases

Eukaryotic RNA Polymerases

• Three DNA dependent RNA polymerases: RNA Pol I, II, and III

• All 3 are big, multimeric proteins (500-700 kD)

• All have 2 large subunits with sequences similar to and ' in E. coli RNA polymerase, so catalytic site may be conserved

• All interact with general transcription factors-GTFs

• RNA Pol II is most sensitive to -amanitin

Page 3: Eukaryotic RNA Polymerases

RNA Polymerase II Inhibitor -Amanitin “The Destroying Angel” - Amanita phalloides

Bicyclic octapeptideBlocks elongation

pol II >> pol III >>> pol I

Page 4: Eukaryotic RNA Polymerases

Yeast RNA Polymerase II Subunits

Page 5: Eukaryotic RNA Polymerases

Transcription Factors

The three RNA polymerases (I, II and III) interact with their promoters via protein:protein and protein:DNA interactions

These proteins or transcription factors (TFs) recognize and initiate transcription at specific promoter sequences

Some transcription factors (TFIIIA and TFIIIC for RNA polymerase III) bind to specific recognition sequences within the coding region

Page 6: Eukaryotic RNA Polymerases

Helix-Turn-Helix Motif

Page 7: Eukaryotic RNA Polymerases

Zinc-Finger Motif: C2H2 Class

Page 8: Eukaryotic RNA Polymerases

Basic Region-Leucine Zipper Motif: bZIP

Page 9: Eukaryotic RNA Polymerases

bZIP Transcription Factor

Page 10: Eukaryotic RNA Polymerases

General Transcription Factors (GTFs)

• GTFs position RNAPs at transcription initiation sites, forming the preinitiation complex (PIC)

• Transcription-initiation complex = RNAP + general transcription factors (GTF) bound to promoter region

• Many of the GTFs that associate with RNAP II initiate transcription from TATA box-containing promoters have been identified– TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH– TFII = “transcription factor RNAP II”

Page 11: Eukaryotic RNA Polymerases

General Transcription Factors

• TFIID is largest and consists of a TATA-box binding protein (TBP) and 8-10 TBP-associated factors (TAFIIs)

• TBP is a “universal transcription factor” – associates with promoters of all three RNAPs, and promoters with and without a TATA box

• Binding of TAFIIs extend the interactions of TFIID

• TFIID has two roles: – foundation for the transcriptional PIC complex– Prevents nucleosome stabilization in the promoter

region (antagonist to H1)

Page 12: Eukaryotic RNA Polymerases

TBP is used by all 3 RNA polymerases

• TBP is a subunit of an important GTF for each of the 3 RNA polymerases:

– TBP or TFIID for Pol II

– SL1 for Pol I

– TFIIIB for Pol III

• It does NOT always bind to TATA boxes; promoters for RNA Pol I and Pol III (and even some for Pol II) do not have TATA boxes, but TBP is still used.

• The GTFs that contain TBP may serve as positioning factors for their respective polymerases.

Page 13: Eukaryotic RNA Polymerases

Yeast TATA Binding Protein: TBPTBP binds in the minor groove of DNA

TBP binding bends the DNA

Page 14: Eukaryotic RNA Polymerases

• RNA Pol I transcribes genes for the large rRNA precursor

• There are hundreds of similar copies of this gene in each genome

• RNA Pol I promoters (called class I) have two components:

• Upstream control element: –156 to -107• Core element: –45 to +20

• Two different transcription factors bind these sequences co-operatively: SL1 and UBF (TBP is a component of SL1)

Promoters for RNAP I

Page 15: Eukaryotic RNA Polymerases
Page 16: Eukaryotic RNA Polymerases

Transcribes various small RNAs: 5S rRNA, tRNA precursors, U6 snRNA, etc

The 5S rRNA and tRNA genes have class III promoters

U6 snRNA and 7SL RNA gene have promoters that resemble RNAP II promoters

The 5S rRNA promoters are entirely within the coding region of the gene

The tRNA promoters contain two elements

The other promoters for U6 snRNA etc appear similar to RNAP II promoters (have TATA boxes) but -amanitin test indicates these are transcribed by RNAP III.

Promoters for RNAP III

Page 17: Eukaryotic RNA Polymerases

PIC Assembly for RNA Pol III Genes

TBP TBP

TBPTBP

Page 18: Eukaryotic RNA Polymerases

RNA Polymerase II General Transcription Factors

Factor Subunits FunctionTFIID - TBP 1 Recognize core promoter (TATA), Recruit TFIIBTFIID - TAFs 12 Recognize core promoter (non-TATA), Pos and Neg

regulatory functions, HAT activityTFIIA 3 Stabilize TFIID and promoter bindingTFIIB 1 Recruit RNA Pol II / TFIIF, Start site selectionTFIIF 2 Assist RNA Pol II to bind promoterRNA Pol II 12 Enzymatic synthesis of RNA, Recruit TFIIETFIIE 2 Recruit TFIIH, Modulate TFIIH helicase, ATPase,

and kinase activitiesTFIIH 9 Promoter melting using helicase, Promoter

clearance via CTD phosphorylation

Modified from Roeder, R.G., Trends in Biochem. Sci. 21:327-334

Page 19: Eukaryotic RNA Polymerases

• Important for helping TBP to bind to promoters that lack TATA boxes.

• There are different TAFs in different cells!

• In vivo these factors are associated with additional proteins forming a larger complex of about 50 polypeptides.

• It is hypothesized that this high MW complex may preassemble and interact with promoters in a single step.

TBP-associated Factors (TAFs or TAFIIs)

Page 20: Eukaryotic RNA Polymerases

TFIID binds TATA box via TBP subunit

TFIIA facilitates and stabilizes binding of TFIID complex

Eukaryotic RNA Pol II TranscriptionFormation of the PIC

Page 21: Eukaryotic RNA Polymerases

TFIIB binds to TFIID

TFIIB is a monomeric protein

C terminal domain contacts DNA and TBP

N terminal domain extends towards start site

Proximity of certain promoter- and enhancer-transcription factors (important for developmental regulation- their activation domain binds directly to TFIIB)

TFIIH close as well at this point, but not yet bound

Eukaryotic RNA Pol II TranscriptionFormation of the PIC

Page 22: Eukaryotic RNA Polymerases

TFIIF binds to RNAPII (preformed complex) – directs RNAPII to promoter

Binding of TFIIE to TFIIF/RNAPII complex and already positioned TFIIB helps positioning the RNAPII over start site:

Two large subunits of RNAP II interact with promoter DNA: CTD tail (unphsphorylated form) of RNAPII is in direct contact with TFIID

TFIIE is DNA-dependent ATPase- probably necessary for generating the energy for transcription

Eukaryotic RNA Pol II TranscriptionFormation of the PIC

Page 23: Eukaryotic RNA Polymerases

Binding of RNAP II/TFIIF/TFIIE to promoter activates TFIIH

TFIIH contains nine subunits

It has helicase activity- unwinds DNA downstream from the initiator site in the presence of ATP necessary for promoter clearance

It has protein kinase activity- phosphorylation of CTD tail of RNAPII

Phosphorylation detaches RNAPII from TFIID

Beginning of transcription by RNAPII

Eukaryotic RNA Pol II TranscriptionFormation of the PIC

Page 24: Eukaryotic RNA Polymerases

Carboxyl-Terminal Domain (CTD Tail)

• Stretch of 7 amino acids that is repeated multiple times (26-52 times): Tyr-Ser-Pro-Thr-Ser-Pro-Ser

• Critical for viability

• CTD tail becomes phosphorylated on ser and some tyr residues as the RNAP transcribes away from the promoter

Page 25: Eukaryotic RNA Polymerases

RNA Pol II PromotersConsist of two parts:

1. Core promoter: - TATA box (position at ~ -30)- initiator (on the transcription start site)

2. Proximal Promoter Elements (can be upstream, downstream or internal)

DownstreamElement

UpstreamElement

TATA Initiator

Page 26: Eukaryotic RNA Polymerases

Eukaryotic TATA Box

ConsensusSequence

TATA motif is usually located at position -25

Page 27: Eukaryotic RNA Polymerases

Eukaryotic Promoter Regions

Initiator

Page 28: Eukaryotic RNA Polymerases

RNA Polymerase II Promoter Consensus Sequences of Transcription Factor Binding Sites

Page 29: Eukaryotic RNA Polymerases

Proximal Control Elements of Genes

Modular Factor Binding Sites

Page 30: Eukaryotic RNA Polymerases

Coordinate Regulation Via Response Elements

Multiple genes are transcribed in response to different cues: for example, heat shock, hormone levels, developmental events, phorbol esters, heavy metals, metabolite concentrations, etc.

Similarly responsive genes will have a DNA sequence located in cis to the gene called a response element.

These response elements are binding sites for transacting factors that are activated in response to the environmental cue.

The location of these elements relative to the start site of transcription is not conserved between genes: eg. a cis element that leads to transcription in response to a hormone may be located at –300 in one gene and –175 in another

Page 31: Eukaryotic RNA Polymerases

Response Elements: Coordinate Regulation

Metallothionein Gene Promoter

Page 32: Eukaryotic RNA Polymerases

Coordinate Regulation by Hormones/Steroid Receptors

Adapted from Molecular Biology of the Cell, 4th Edition

Page 33: Eukaryotic RNA Polymerases

Steroid Receptors

Upon binding to the hormone cortisol, the cytoplasmic glucocorticoid receptor displaces an inhibitory protein (Hsp90) and moves to the nucleus where is can interact with glucocorticoid response elements (GREs) in the DNA affecting gene transcription.

Adapted from Molecular Biology of the Cell, 4th Edition

Page 34: Eukaryotic RNA Polymerases

Activation of Transacting Factors

Page 35: Eukaryotic RNA Polymerases

Control of Cellular Differentiation By TFs

Page 36: Eukaryotic RNA Polymerases

Enhancers

Control elements that stimulate transcriptionBind multiple different transcription factorsTranscription factors that recognize enhancer =

activators or enhancer binding proteinsActivators interact with general transcription factorsNegative enhancer is a silencer

Page 37: Eukaryotic RNA Polymerases

Stimulate expression of genes over long

disances (up to 50kb)Occur upstream, downstream, in introns

or in exonsOrientation independentMay be cell-type specific

Enhancers

Page 38: Eukaryotic RNA Polymerases

Activation of transcription initiation in eukaryotes by recruitment of the eukaryotic RNA polymerase II holoenzyme complex (100 protein subunits).

Enhancers: Action at a Distance

Page 39: Eukaryotic RNA Polymerases

Insulators / Boundary Elements

Boundary elements block encroachment of heterochromatin from neighboring loci. They also stop the unregulated enhancement or activation of neighboring genes outside of their chromosomal domain.

Page 40: Eukaryotic RNA Polymerases

Hypomethylation(Active)

Vs.Hypermethylation

(Silenced)

Page 41: Eukaryotic RNA Polymerases

Silencing: Histone Deacetylation HDAC

• Some repressors recruit histone deacetylase, which removes acetyl groups from histones resulting in gene silencing

Page 42: Eukaryotic RNA Polymerases

Histone Acetylation and Deacetylation

AcCoA

C

O

CHNHCH2

C

O

NH... ...CH2

CH2

CH2

CH2

NH 3+

C

O

CHNHCH2

C

O

NH... ...CH2

CH2

CH2

CH2

NH

Gly Lys

CCH3

O

CoA

AcPositive charge on amino group No charge on amide group

Histone acetyl transferases

Histone deacetylases

HAT

HDAC

Page 43: Eukaryotic RNA Polymerases

Histone tails are postranslationally modified by acetylation, methylation, phosphorylation and ubiquitination. These modifications have a profound effect on gene activity. The specific set of modifications is termed the “histone code” .

Histone Code

Page 44: Eukaryotic RNA Polymerases

Remodeling complexes allow access of replication and transcription factors to the DNA.

Remodeling requires ATP

Chromatin Remodeling

Page 45: Eukaryotic RNA Polymerases

Gene Activation By Chromatin Remodeling

Transcription Factor Access

Page 46: Eukaryotic RNA Polymerases

HATs open chromatin

Nucleosome remodelers

Transcription Factor Coactivators

Page 47: Eukaryotic RNA Polymerases

Assembly of preinitiation complex on

open chromatin

Page 48: Eukaryotic RNA Polymerases

Chromatin remodeling of diploid somatic cell nucleus in the egg cytoplasm reprograms the nucleus to recapitulate development.