Post on 01-Jan-2016
transcript
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