General Transcription Initiation Factors

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General Transcription Initiation Factors
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RNA polymerase II underlies the central dogma of
molecular biology
Pol I
rRNA
Pol II
Ribosome
DNA
mRNA
Protein
Pol III
tRNA
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RNA polymerase II transcription machinery
DNA unwinding RNA polymerization proofreading
Pol II
promoter recognition
GTFs (TFIIB, -D, -E, -F, -H)
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Conserved Mediator subunits
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Mediator of Transcriptional Regulation

• Required for all transcription of all pol II
  promoters
• Essential link in the chain of communication
  enhancer activator Mediator pol II
  promoter
• Co-activator, co-repressor, and general
  transcription factor
• Processes, transduces transcriptional regulatory
  information in all eukaryotes

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Accurate initiation by RNA polymerase II plus
factors in the nucleus
Eukaryotic RNA pol II

Nuclear extract, S-100

Promoter
General Transcription Initiation Factors
initiation
Nascent RNA
elongation
Run-off transcript, Discrete size


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General transcription factors GTFs

• Proteins other than RNA polymerase involved in
  transcription
• Initiation, Elongation, Termination
• General transcription initiation factors (GTFs)
• Proteins required for specific transcription from
  a minimal promoter (core)
• Not subunits of purified RNA polymerase.
• Required for RNA polymerase to bind avidly and
  specifically to promoters.
• GTIFs for RNA polymerase II are called TFIIx,
  where x A, B, D,
• Can have multiple subunits

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Fractionation of nuclear extracts to find
GTFsPhosphocellulose column ion exchange
Plus Purified Pol II
Run-off Transcript Accurate Initiation at
promoter
Template Adenovirus late promoter
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Fractionation of nuclear extracts to find
GTFsFractionation scheme, DEAE cellulose
Plus Purified Pol II
Run-off Transcript Accurate Initiation at
promoter
Matsui, Segall, Weil, Roeder (1980) JBC 25511992
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The basal machinery
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GTFs for RNA polymerase II
Modulates helicase
Helicase
helicase
IIF
IIH
Targets Pol II to promoter
Recognize core promoter
CTD protein kinase
protein kinase
IIF
IIH
CTD of large subunit of Pol II
Many GTFs are possible targets for activators of
transcription.
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The TATA-binding protein
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TBP (Tata-binding protein) binds to the TATA box
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TFIIB, TFIIE, TFIIF

• TFIIB
• Binds after TBP
• Mutants can affect start site selection
• TFIIF
• RNA polymerase II-associated (Greenblatt)
• Two subunits, RAP30 and RAP74
• RAP74 thought to play a role in elongation
• On partially open templates, transcription can
  occur
• with RAP30, TFIIB and TBP
• TFIIE
• Two subunits
• Plays a role after recruitment of pol II
• May function with TFIIH in promoter escape

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The TBP/TFIIB/TFIIA complex
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TBP/B/A from different angle
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TFIIH
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2-D protein crystallization

• rapid, small amount of protein (mg)
• pol II without subunits 4 and 7
• 2-D crystals seed 3-D crystal growth

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Obstacles to X-ray structure determination of pol
II
Oxidation Crystal polymorphism No standard
heavy atom derivatives
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Structure determination of RNA polymerase II

• 1971 Lateral diffusion in lipid layers
• 1983 2-D protein crystals on lipid layers
• 1991 2-D crystals seed 3-D crystals
• Diffraction phased with heavy
• atom clusters
• 2000 Structure at 2.8 Å resolution

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10 years, 10,000 liters, 1 grad student
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Proteins 10 Amino acids 3559 Atoms 28,378
MW 0.5 MDa Rfree (2.8 Å) 28.2
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Pol II
Nontemplate
Template
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Hybrid
RNA
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NTP rotates into A site
pre-translocation
post-translocation
NTP enters E site
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Trigger loop contacts NTP in the A site
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Multiple conformations of the trigger loop
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Trigger Loop Network
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Trigger Loop Network
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Trigger loop couples nucleotide selection to
catalysis
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Nucleotide selection by alignment with the
trigger loop, coupling recognition to catalysis
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Trigger Loop Network
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F
Pol
H
E
TBP
BC
4/7
BN
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F
Pol
H
E
TBP
BC
4/7
BN
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Pol
H
E
TBP
BC
F
BN
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Pol
H
E
TBP
BN
BC
F
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Pol
H
E
BC
TBP
F
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E
Pol
H
BC
TBP
F
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Complete minimal RNA polymerase II transcription
initiation complex
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Mechanism of initiation of RNA polymerase
II transcription
Pol
Pol
H
H
F
BN
BC
BC
TBP
TBP
Factor
Role
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Pol II Elongation

• DNA melting
• Nucleotide selection
• DNA/RNA separation
• Polymerase translocation

Template Non template RNA
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Pre-translocation (pdb 1I6H)
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Wall Dock Clamp
Template Non template Active site Mg2 RNA
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Post-translocation (pdb 1SFO)
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Two Site Model
Active site
Non-specific site
Template Non template Active site Mg2 RNA
NTPs enter via pore