Transcript RNA polymerases #2 - Pennsylvania State University

General Transcription Initiation
Factors
Assay for accurate initiation by bacterial RNA polymerase


E. coli RNA pol
holoenzyme


+
Promoter
initiation




Nascent RNA
elongation


Run-off transcript,
Discrete size

RNA Pol core
'

+
+


'
Random transcription by bacterial RNA
polymerase core

E. coli RNA pol core


Promoter

+



'
Random initiation

'
'


'
Transcripts of random size


elongation


'




'
Random transcription by eukaryotic RNA polymerase
Eukaryotic RNA pol II
Promoter
+
Random initiation
elongation
Transcripts of random size
Accurate initiation by Euk RNA polymerase II plus
factors in the nucleus
Eukaryotic RNA pol II
Promoter
+
Nuclear extract, S-100
+
General
Transcription
Initiation
Factors
initiation
Nascent RNA
elongation
Run-off transcript,
Discrete size
+
+
General transcription factors = GTFs
• Proteins other than RNA polymerase involved in
transcription
– Initiation, Elongation, Termination
– Not subunits of purified RNA polymerase.
• GTFs for RNA polymerase II are called TFIIx,
where x = A, B, D, …
– Can have multiple subunits
• General transcription initiation factors (GTIFs)
– Proteins required for specific transcription from
a minimal promoter (core)
– Required for RNA polymerase to bind avidly and
specifically to promoters.
Fractionation of nuclear extracts to find GTFs
Phosphocellulose column: ion exchange
Plus
Purified
Pol II
Run-off
Transcript:
Accurate
Initiation at
promoter
Template: Adenovirus late promoter
Polyacrylamide gel separating
products of in vitro transcription
Fractionation of nuclear extracts to find GTFs
Fractionation scheme, DEAE cellulose
Plus
Purified
Pol II
Run-off
Transcript
Accurate
Initiation at
promoter
Matsui, Segall, Weil, Roeder (1980) JBC 255:11992
GTIFs for RNA polymerase II
Modulates helicase
IIA
IIB
TAFs
TBP
IIE
}
IIH
Targets Pol II
to promoter
IIA
protein kinase
CTD protein
kinase
IIF
IIE
IIB
IIF
TFIID
Recognize core promoter
Helicase
helicase
Pol IIa
Inr
IIH
TBP
CTD of large subunit of Pol II
Many GTIFs are possible targets for activators of transcription.
TATA Binding Protein = TBP
• TBP binds in the narrow groove of DNA at
the TATA box found about 20-25 bp 5’ to the
start site for transcription of many (but not
all) genes transcribed by RNA polymerase
II.
• TBP bends the DNA about 90 degrees.
• TBP alone or with TBP-associated proteins
(TAFs) plays an important role in
recognizing the core promoter and recruiting
RNA polymerase II to the promoter.
TBP bound to DNA
Image from crystal structures, provided by Dr. T. Nixon.
RNA Pol II bound to DNA and general
transcription initiation factors
RNA polymerase II f rom yeast
o
100 A thick
(does not include subunit s 4 and 7)
o
16 A resolut ion
o
140 A
DNA
o
136 A
f lexible end of "thumb"
TFIIE
o
Channel 25 A diamet er
o
Groove 25A wide
TFIIA
TFIIB
TBP
Channel plus groov e could accomodate
20-25 bp of duplex DNA
Dar st, S.A., A.M. Edwar ds, E.W. Kubalek & R.D. Kornberg
(1991) Cell 66: 121-128.
Kor nber g, R.D. ( 1996) Trends in Bioch. Sci. 21: 325- 326.
Movie of TBP, then binding to TATA
DNA (and bending it), and then TFIIA
binding to TBP-TATA
Thanks to Drs. Song Tan and
Tracy Nixon for movies
Sequential
Binding
Model for
assembly
of
preinitiation
complex
-30
+1
TATA
Inr
TAFs
}
TBP
TFIID
or TBP
IIA
IIB
Eukaryotic RNA
polymerase II
IIF
Pol IIa
CTD of large subunit of Pol II
IIE
helicase
IIH
protein kinase
IIF
IIE
IIA
IIB
TATA
Inr
Pol IIa
IIH
preinitiation complex
= PIC
ATP hydrolysis
IIE
Polymerization of 1st few NTPs and
phosphorylation of CTD leads to promoter
clearance. TFIIB, TFIIE and TFIIH
dissociate, PolII+IIF elongates, and TFIID +
TFIIA stays at TATA.
IIA
IIB
TATA
IIF
Pol IIa
IIH
Inr
initiation complex, DNA melted at Inr
Activated
PIC
Direct
Binding of
Holoenzyme
model for
assembly of
the
preinitiation
complex
-30
+1
TATA
Inr
TBP
TAFs
or
TBP
TFIID
Inr
TATA
IIA ?
}
SRB
IIF
IIE
IIB
Holoenzyme
Pol IIa
IIH
activator
?
SRB
IIF
IIE
IIB
IIA
TATA
preinitiation complex
Pol IIa
Inr
= PIC
IIH
ATP hydrolysis
SRB
IIE
IIF
Activated
PIC
initiation complex,
DNA melted at Inr
IIB
IIA
TATA
Pol IIa
IIH
Other proteins involved in transcription
and regulation
• In addition to RNA polymerase II and GTIFs:
• Proteins required for regulation, e.g.
– Gal11: regulation of the GAL operon
– Rgr1: resistance to glucose repression
• Srb proteins
– Yeast strains with truncations in the CTD of the large
subunit of RNA polymerase B are cold-sensitive
– SRB genes: when mutated, suppress the phenotype of
CTD deletions
– Extragenic suppressors: implicated in RNA polymerase
function
RNA polymerase II Holoenzyme and Mediator
• Holoenzyme
– RNA polymerase II + (TFIIB, E, F, H )+ (Srb2, 4, 5, 6) +
(Rgr1, Gal11, others)
– Correct initiation in presence of TBP (TFIID)
– Responds to transcriptional activators
• Mediator
– Complex needed for a response to transcriptional
activators by purified RNA Pol II plus GTFs
– Yeast Mediator has 20 subunits, including Srb2, 4, 5, 6;
Srb7, Rgr1, Gal11, Med 1, 2, 6, 7, Pgd1, Nut 1, 2, and
others
• RNA Pol II + Mediator (+ some GTIFs?) =
Holoenzyme
Expanding the functions of RNA
polymerase
Polymerase
Transcribe
RNA Pol II
Yes
RNA Pol II + Yes
GTIFs
RNA Pol II
Yes
holoenzyme
+ GTIFs
Start at
Promoter
No
Yes
Respond to
Activator
No
No
Yes
Yes
Parallels
between
initiation
pathway in
prokaryotes and
eukaryotes
From Eick et al. (1994)
Trends in Genetics 10: 292-296
T AFs
T BP
}
Pol IIa
T FIID
polym erase
GTFs for RNA polymerase I
Pol I
Eukaryotic RNA polymerase I
UBF1
}
SL1
T BP
Pol III
Eukaryotic RNA polymerase III
}
T BP
IIIC
T FIIIB
IIIA
IIIC
UBF1
GTFs for RNA}polymerase III
SL1
T BP
Pol III
Eukaryotic RNA polymerase III
}
T BP
IIIC
T FIIIB
IIIA
IIIC