Eukaryotic RNA Polymerases
description
Transcript of Eukaryotic RNA Polymerases
Gene Expression
Eukaryotic Gene Transcription9/18/08
Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics
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
RNA Polymerase II Inhibitor -Amanitin “The Destroying Angel” - Amanita phalloides
Bicyclic octapeptideBlocks elongation
pol II >> pol III >>> pol I
Yeast RNA Polymerase II Subunits
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
Helix-Turn-Helix Motif
Zinc-Finger Motif: C2H2 Class
Basic Region-Leucine Zipper Motif: bZIP
bZIP Transcription Factor
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”
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)
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.
Yeast TATA Binding Protein: TBPTBP binds in the minor groove of DNA
TBP binding bends the DNA
• 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
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
PIC Assembly for RNA Pol III Genes
TBP TBP
TBPTBP
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
• 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)
TFIID binds TATA box via TBP subunit
TFIIA facilitates and stabilizes binding of TFIID complex
Eukaryotic RNA Pol II TranscriptionFormation of the PIC
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
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
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
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
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
Eukaryotic TATA Box
ConsensusSequence
TATA motif is usually located at position -25
Eukaryotic Promoter Regions
Initiator
RNA Polymerase II Promoter Consensus Sequences of Transcription Factor Binding Sites
Proximal Control Elements of Genes
Modular Factor Binding Sites
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
Response Elements: Coordinate Regulation
Metallothionein Gene Promoter
Coordinate Regulation by Hormones/Steroid Receptors
Adapted from Molecular Biology of the Cell, 4th Edition
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
Activation of Transacting Factors
Control of Cellular Differentiation By TFs
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
Stimulate expression of genes over long
disances (up to 50kb)Occur upstream, downstream, in introns
or in exonsOrientation independentMay be cell-type specific
Enhancers
Activation of transcription initiation in eukaryotes by recruitment of the eukaryotic RNA polymerase II holoenzyme complex (100 protein subunits).
Enhancers: Action at a Distance
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.
Hypomethylation(Active)
Vs.Hypermethylation
(Silenced)
Silencing: Histone Deacetylation HDAC
• Some repressors recruit histone deacetylase, which removes acetyl groups from histones resulting in gene silencing
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
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
Remodeling complexes allow access of replication and transcription factors to the DNA.
Remodeling requires ATP
Chromatin Remodeling
Gene Activation By Chromatin Remodeling
Transcription Factor Access
HATs open chromatin
Nucleosome remodelers
Transcription Factor Coactivators
Assembly of preinitiation complex on
open chromatin
Chromatin remodeling of diploid somatic cell nucleus in the egg cytoplasm reprograms the nucleus to recapitulate development.