General Transcription Factors: GTFs, TBP/TFIID and others
TBP Associated Factors: TAFs
Elongation of Transcription (??)
Transcription Regulation and Gene Expression in Eukaryotes FS 08
Basal Transcription (2)
P. Matthias, March 05, 2008
TFIIB
RNAPII
TFIIATFIID
TFIIH TFIIFTFIIE
The Players in Transcription Regulation• DNA-binding transcription factors (upstream factors)• Chromatin regulators• Coactivators and corepressors: Mediator, etc..• Basal Machinery: RNA PolII, GTFs
Gene
BasalMachinery
DNA-binding TFs
Chromatin regulatorsMediator
Coactivator
Factors needed for expression of nearly all Class II promoters
Biochemical Fractionation (R. Roeder, Rockefeller University) led to identification of 6 fractions that contain proteins that permit correct transcription initiation by RNA pol II
These fractions were labeled TF II: A, B, D, E, F, H. (Fraction C as an artefact.)
Of these TFIID, TFIIA and TFIIB bind specific proximal elements (others such as TFIIH bind DNA but are not thought to have strict DNA sequence preference)
TFIID nucleates the assembly of the pre-initation complex (PIC)
GTFs: recognize the core promoter
TATA INR
core
GENE
upstream(regulatory,enhancer)
Basalmachinery
RNA PolII 12TFIIA 3TFIIB 1TFIID (TBP) ca. 14 (1)TFIIE 2TFIIF 2TFIIH ca. 9
Transcription activatorsNF-kB
FosOct-1CBFA-1CREBetc…
Eukaryotic Pol II promoter: upstream and core
PyPyANT/APyPy GA/TCGTGTATAAAG/CG/CG/CCGCC
DPEInitiatorTATA boxBRE
+31-32 -26 +1
The core promoters in more detail
TAFII250TAFII150
TAFII60TAFII40
TBP
TFIIB TFIID TFIIDTFIID
TFIIA
TATA-less
TATA-containing
In vitro Assembly of GTFs + Pol II
Ordered assembly of the Pre-Initiation Complex (PIC)
• D - A - B -F/PolII - E - H
Assembly of the Basal Machinery
Alternative Models for Assembly of the PIC
PolII Holoenzyme
•How to measure PIC assembly in vivo?
TFIID
• TFIID is a multiprotein complex: TBP + TAFs– (~10-14 different subunits)
• The subunit responsible for recognizing the TATA box is called ‘TATA-Binding Protein’ (‘TBP’)
• TBP can specifically bind to TATA-boxes on its own
• In vitro TBP is sufficient for “basal” transcription, not for “activated”transcription
TAFIIs of TFIID:-8 mostly conserved proteins ranging from 30 250 kDa
TBP: a GTF essential for all polymerases
TBP-related factors
TBP-related factors
TBP-related factors specific complexes
Promoters with TBP and TRFs
TFIIH
• TFIIH is a multiprotein complex required for both transcription and DNA repair
• Human TFIIH is composed of nine polypeptides with a total molecular weight of 460 kDa
• TFIIH contains three ATP-dependent catalytic activities– kinase activity that phosphorylates the CTD (C-terminal domain) of the
largest subunit of Pol II, a – DNA-dependent ATPase– DNA helicase
TFIIH Activities
• TFIIH is responsible for three critical functions in transcription– phosphorylation of the COOH-terminal domain (CTD) of the RPB1
subunit of RNA polymerase II (RNAPII)– promoter melting– promoter clearance.
• Two biochemically separable complexes:– core TFIIH– Cdk Activating Kinase (CAK) complex
Properties of TFIIH
• TFIIH alters the mobility of the PIC complex in the presence of ATP
RNAP making short RNAs, its stalled at +10 - +12.
TFIIH causes further DNA unwinding, allowing the bubble to grow and RNAP to go to elongation phase.
TFIIF
• TFIIF binds to RNAPII in solution and facilitates delivery of the polymerase to the TFIID-TFIIB-DNA complex on the promoter
•• Human TFIIF contains two separate subunits
– RAP76– RAP34
• TFIIF is required for entry of TFIIE and TFIIH into the pre-initiation complex
TFIIE
• The general transcription factor TFIIE plays important roles at two distinct but sequential steps– open complex formation– transition from initiation to elongation.
• The large subunit of human TFIIE (TFIIE ) binds to and facilitates the enzymatic functions of TFIIH, but TFIIE also functions independently fromTFIIH
• Human TFIIE consists of two subunits, TFIIEa (57 kDa) and TFIIEb (34 kDa)
• The subunits combine into a α2β2 heterotetramer with a combined molecular mass of 180 kDa
• TFIIE plays essential roles in the regulation of TFIIH activities– CTD kinase and ATPase are positively regulated, DNA helicase activity is
negatively regulated
TFIIDTBP }TAFsIIB
IIA
IIE
IIF
IIH
helicase
protein kinase
TBPInr
IIB IIA Pol IIa
IIFIIE
IIH
CTD of large subunit of Pol II
Recognize core promoterTargets Pol II to promoter
Modulates helicase
Helicase
CTD protein kinase
Summary
What are the in vivo targets of transcription activators???
East coast vs West coast,Yeast vs Metazoans,In vivo vs in vitro, ...
GTFs
TBP
Sp1 +TFIID
+
TBP vs TFIID: TAFs as universal coactivators??
Albright and Tjian (2000). TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 1-13.
Albright and Tjian (2000). TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 1-13.
Beyond TFIID: Multiple TAF complexes
Beyond TFIID: Multiple TAF complexes
Which part of the promoter is TAF responsive?
Unified TAF nomenclature
In vivo rates of transcription = 1200-2000 nucl/min.
Baseline in vitro rates = 100-300 nucleotides/min.frequent pausing and arrests even with chromatin-free DNA
Large number of proteins and protein complexes thatregulate elongation of transcription
•Suppress pausing •Promote pausing•Overcome transcription arrest•Enable elongation through chromatin
Transcription Elongation
•Suppress pausing and/or arrestTFIIFP-TEFb (CTD kinase)S II (TFIIS)CSB (Cockayne syndrome binding pro.)ELL 1/2, Elonginscertain transcription activators (e.g. heat shock factor)
•Promotes pausing/arrestDSIF (DRB sensitivity inducing factor)NELF (binds early during RNA elongation)
•Enable elongation through chromatinFACTRSFElongator (HAT associated with Pol II)
Elongation factors for RNA Pol II
NELF and DSIF promote arrest of unphosphorylated RNA polymerase.
P-TEFb phosphorylates CTD and Spt4/5 Release of NELF and DSIF inhibited
Annu. Rev. Biochem. 1999. 68:301-319
Elongation regulation can be a target in human diseaseHIV - P-TEFb works with HIV TAT protein to promote HIV RNA transcription
Acute Myeloid Leukemia - ELL gene is fused in chromosomal translocation to MLL gene
Cockayne Syndrome - DNA repair deficiency disease CSBprotein mutated in multiple cancers
Polyglutamine Diseases
• Several fatal neurodegenerative diseases are due to expanded polyglutamine tracts present in some proteins
– Includes Huntington’s disease (HD), Kennedy’s disease, spinocerebellar ataxias (SCAs)
– The number of CAG repeats is 10 to 34 in normal individuals and 37 to 100 in HD patients
• Progressive loss of motor and cognitive functions due to neuronal degeneration in the central nervous system
Molecular Effects of polyQ Expansions
• Proteins containing expanded polyglutamine tracts form insoluble aggregates and inclusion bodies
• Expanded polyglutamine tracts appear sufficient for the neurotoxic phenotype (i.e., they do not have to be associated with any specific protein!)
• Transcriptional dysregulation detectable before onset of symptoms– Yeast and mouse model systems
• Interaction between expanded polyglutamines and HAT-CREB-binding proteins?
TBP and Disease: Pathology of TBP Mutations
• N-terminal domain contains polyglutamine repeat encoded by ‘CAG’ codons
• Many types of ‘polyglutamine’ expansions are linked causally to neurodegenerative diseases
• Abnormal expansion of glutamine repeat in human TBPcauses dominant cerebellar ataxia
Inverse Correlation Between Number of CAGRepeats and Onset of Disease
Nakamura et al. (2001). SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein. Hum. Mol. Genet. 10, 1441-1448.
PCR Immunoblot Analysis of SCA17
Nakamura et al. (2001). SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein. Hum. Mol. Genet. 10, 1441-1448.
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