Three RNA polymerases in eukaryotes

Three RNA polymerases in eukaryotes

RNA polymerase III

Hundreds of promoters - 40% of a cell transcriptional activity-Moderately sensitive to a-amanitin (Ki=1mM)-Transcribe gene encoding tRNAs, 5S rRNA and some stable RNAs (U6 and others)-Promoter may be internal to the transcribed region

RNA polymerase II

Thousands of promoters - 10% of a cell transcriptional activity-Highly sensitive to a-amanitin (Ki=10 nM)

Transcribe gene encoding mRNAs and some stable non coding RNAs (U1, U2, U4, U5 and others)

RNA polymerase I

1 promoter - 50% of a cell transcriptional activity- low sensitivity to a-amanitin

Transcribe gene encoding the 45S precursor of rRNAs (18S, 5.8S, 28/26S rRNAs)

18S 28S 28S18S5.8S 5.8S

45S precursor

Non-transcribed spacer

45S precursor

TATA Inr

TATA Inr

TATA Inr

TATA Inr

TATA Inr

+ TFIID+ TFIIA

+ TFIIB

+ TFIIF - Pol.II

+ TFIIE+ TFIIH

Pre InitiationComplex (PIC)

IIA

IIA

IIA

IIA

TFIID(TBP +TAFs)

TFIID

TFIID

TFIID

TFIIB

IIB

IIB

TFIIF

RNA Polymerase II

Polymerase II

TFIIFTFIIE

TFIIH

Assembly of the RNA polymerase II machinery

onto Eukaryotic Promoters

General Transcription Factors: used to assist RNA Polymerase bindingto most promoters:TFIIATFIIBTFIID = TBP +TAFsTFIIETFIIH

TATA Inr

ATP hydrolysis(helicase in TFIIH)Also used in Nucleotide Excision Repair

Pre InitiationComplex (PIC)

OpenComplex

IIATFIID IIB

Polymerase II

TFIIFTFIIE

TFIIH

TATA InrIIA

TFIID IIBPol. II

TFIIFTFIIE

TFIIH

TATA InrIIA

TFIID IIBPol. II

TFIIFTFIIE

TFIIH

PP P

P

Pol. II CTDPhosphorylationby CAK (TFIIH)

Phosphorylation at S5 (initiation)of YSPTSPS repeats of the CTD

TATA InrIIA

TFIID IIBPol. II

TFIIFTFIIE

TFIIH

PP P

P

TATA InrIIA

TFIID IIBPol. II

TFIIFTFIIE

TFIIH

PP P

P

CappingEnzyme

SRProteins

CPSFCstF

Phosphorylated Pol. II

Recruitment of RNA processing Factors

Capping enzyme,SRs (splicing)CPSF, CstF (3’end)

TranscriptionInitiation

TATA InrIIA

TFIID Pol. II

TFIIFTFIIE

TFIIHTFIIB

CappingEnzyme

SRProteins

CPSFPP P

P5’

3’

Phosphorylation at S2 (elongation)

of YSPTSPS repeats of the CTD

TATA Binding Protein (TBP)

- binds and recognize the TATA box

TFIID

TBP-associated Factors (TAFs)TAF 250,60, 110, 95, 78, 38, 28

- binds and recognize the Inr (TAF250)

-provide binding sites for gene specifictranscription factors

TFIIHXPB ATP-dependent DNA helicasep62p52p44P34

XPD ATP-dependent DNA helicaseCyclin-activated kinase:Phosphorylates the CTD of Pol. II

Structure of TBP bound to the TATA element (coding strand shown in green)

Composition and Function of two GTFs – TFIID and TFIIH

PDB ID = 1CDW

Transcriptional Control in eukaryotes:a few things specific to eukaryotes

-------------------------------------------------------------------------------------------• Gene-specific transcription factors(as opposed to sigma factors)

• Coactivators of transcription

• Transcriptional control and chromatin modifications(“epigenetics”)

(control of transcription by controlling RNA Pol.II binding)-------------------------------------------------------------------------------------------• Pausing of the RNA polymerase near promoters

(control of transcription by controlling RNA Pol.II elongation)

TATA box InrActivatorSequence(sometimes called enhancer)

+1-30

GTFs + Pol. IIGene-specifictranscription factor

binds here

Promoter

Why Are activator sequences necessary ?

Transcriptional Activation in Eukaryotes: Genes-specific transcription factors

1) Assembly of GTFs and Pol. II is inefficient The binding of gene-specific TFs facilitate assembly of GTFs and Pol. II 2) Transcription is cell- or time-specific

The presence of a combination of gene specific activators in a particular cell type at a particular stage of differentiation ensures the transcription of the proper set of genes.

Modular Structure of Gene-Specific Transcription Factors

N C

DNA-binding Domain

ActivatorDomain

Examples of Activator Domains:- Acidic- Glutamine-rich- Proline-rich

Examples of DNA-binding Domains: - Helix Turn Helix- Zn Finger- leucine Zippers/bZip- bHLH

Flexible

Linker

TATA box Inr

+1

GTFs+ Pol. II

binding site 1

ActivatorDomain-2 Activator

Domain-1

DNA BindingDomain-2

DNA BindingDomain-1

Enhancer 2

How do long distance (enhancer-promoter)relationships work ?

Local CurvatureOf the DNA region

=> PIC Stabilization

Helix-turn-Helix DNA binding domains

• most frequent DBDin prokaryotes (e.g. Lac Repressor,etc..)

• Also found in euk.for example in Homeodomain proteins = Transcription Factorsthat govern DevelopmentEngrailed, Bithoraxetc…

Courey Plate 4.4 PDB ID = 1HDD

3 Zn Fingerscomplexedto DNA

2 Zn Fingers

The Zn Finger motif1 Zn Finger3D structure

The multiplicityof Zn Fingers

on the same proteinallows recognition

of complex DNAsequences

PDB ID = 1ZAA

Direct base readout by a-helices of DNA binding domains

bZip domains

Helix-loop-Helixdomains

PDB ID: 1FOS

PDB ID: 1HDD

Courey Plate 4.4

TATA box

Coactivator

Inr

Gene-SpecificTranscriptionFactor

GTFs + Pol.II

TATA box

Inr

Gene-SpecificTranscriptionFactor

GTFs + Pol.II

Examples of Coactivators: -CBP/p300-Mediator Complex

Direct vs. Indirect Activation by Gene-Specific Transcription Factors

• Direct Activation: The Gene-Specific Transcription Factor interactsdirectly with the GTFs and/or RNA Polymerase II

• Indirect Activation: The Gene-Specific Transcription Factor does not interactdirectly with the GTFs and/or RNA Polymerase II and needs a Coactivator

- The Mediator complex stimulates transcription of genescontaining activator sequences.

- The action of the Mediator complex is dependent on the presence of proteins binding to the activator sequences.

TATA Inr

TATA InrEnhancer

mediator

From Boyer et al., Nature 1999 May 20;399(6733):276-9

RNA produced by“basal” transcription

RNA produced by“activated” transcription

Conclusion: The presence of the mediator complex only affects RNA produced by “activated” transcription, not by “basal” transcription

One Example of Coactivator complex:The Mediator

In vitro transcription with two different DNA templates, RNA Pol.II, and increasing amounts of mediator complex

Binding of histones to DNA through electrostatic interactions:Histones are + charged, DNA is - charged

-Modulation of interactions of histones with DNA by covalent modifications:Histones acetylation, deacetylation, methylation, ubiquitination on the N-terminal tails of histones

Structure of the nucleosome of eukaryotic cells

The problem of Chromatin in Eukaryotic CellsHow to access genes in this context ??

Post-translational modifications of histonesmodulate chromatin accessibility and transcription

Current Opinion in Plant Biology Volume 5, Issue 5 , 1 October 2002, Pages 437-443

NH3

Ac-CoA

CoA

Lys side chain in a histone tail

HAT

HAT = histone acetyl transferaseHDAC = histone deacetylase

CH3

HN

O

H2O

CH3COO-

HDAC

Histone modifications modulate chromatin accessibility and transcriptional status

• Regulated chromatin modifications,allow access of the transcription machinery and activation/inactivation of genes

MethylTransferase

overall Histone H3acetylation/methylationstatus controlstranscription levels

-> Enhancer Sequences

-> Promoters

-> Transcribed Regions

Hum. Mol. Genet. (2009) 18 (R2): R195-R201.

Specific Histone modifications mark gene regions in eukaryotes“Chromatin Signature”

Chromatin immunoprecipitation of RNA pol.IIacross genomes reveal the enrichment of the

polymerase nearby promoters (“poised”)

Transcriptional Control by Controling RNA Polymerase II elongation

Promoter proximal pausing is controlled by the elongation factor NELF and helps control

eukaryotic gene expression by rapid switch from poised state to elongation