RNA POLYMERASES

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RNA POLYMERASES BCH 6415 SPRING 2006 DR. YANG
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Rate of transcription Euk RNA pol II 15-30
nt/sec Bacterial RNA pol 50-100 nt/sec
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Eukaryotic RNA Polymerase II 12 subunits 10
polypeptide catalytic core 2 subunits (Rpb4/7)
required for transcrip. initiation
Bacterial RNA Polymerase Core enzyme a2ßß?
400dDa (Transcription elongation) Holoenzyme
Core s (Transcription initiation)
Bacterial s subunit homologous to Tfg2 subunit
of eukaryotic GTF TFIIF
fr. Ebright JMB 2000
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Interactions of E. coli RNA pol with promoters
A) Simple promoters a-CTD makes no specific
interactions B) Promoter with upstream element
near or further upstream of -35 contact of a-CTD
with DNA only or with DNA s C) Promoter
with adjacent or upstream DNA-binding activator
that interacts with a-CTD
fr. Chen et al. Molec Cell 2003
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Transcriptional Initiation Bacterial RNA
polymerase-promoter interactions
Sequence-specific interactions via sigma subunit
Fr. deHaseth et al. J Bact. 1998
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The Transcription Cycle
Fr. Mooney et al. J. Bact. 1803265 1998
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Transcription Elongation Complex
(Or could rNTPs enter active site pre-bound
to DNA template?)
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Fr. Mooney et al. J. Bact. 1803265 1998
Diagram of transcription elongation complex (TEC)
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Thermus aquaticus Core RNA Polymerase
ß Blue, Green
a, ? - White
Mg at active site
ß Pink, Green
Green Evolutionarily conserved a.a. seq.
among prokaryotes, eukaryotes
archeaebacteria
Fr. Darst Curr Opin Struc Biol 11155 2001
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Structure of Thermus aquaticus Core RNA Polymerase
Jeruzalmi Steitz EMBO J 1998
Stereo image
Fr. Zhang et al. Cell 1999
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Elongating core
Holoenzyme
Open complex
T. aq. holoenzyme structure
Fr. Young et al. Cell 109417 2002
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Cutaway view of yeast RNA polymerase II
elongation complex
Structure of DNA in the yeast EC
Fr. Klug Science 2921844 2001 Armache et al.
Curr Opin Struc Biol 2005
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Structure of Bacterial RNA Pol Holoenzyme RNAP
Promoter DNA
All sequence-specific contacts with promoter
(e.g., at -10 and -35) are mediated by s
(shown as cylinders)
Fr. Murakami Darst Current Opin Struc Biol 2003
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Larger bubble (fr. -11 to 4) Mg stabilizes
open conformation
Extended footprint
Conformation change Jaws closing?
DNA melting
C closed complex O open complex R RNA pol P
promoter DNA
Abortive initiation
Summary of intermediates in process of
transcription initiation
Fr. deHaseth et al. J. Bact. 1803019 1998
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(Analogous to bacterial closed complex)
(factors left behind at promoter after 1st round
of transcription initiation to facilitate reinitia
tion)
The Eukaryotic RNA Pol II Transcription Cycle
Fr. Hahn Nature Struc Molec Biol. 2004
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-35 element
at AT-rich -10 element
Structural changes in RNAP-DNA from initiation
-- elongation
DNA melting facilitates DNA bending
Cross-section view thru RNAP Dk green
template Lt green nontemplate Orange - s
subunit
Full transcription bubble
Nascent RNA reaches 12 nucleotides and displaces
s3.2 loop RNA exit channel opens Displacement
of s3.2 loop may also destabilize s inteactions
w/ core facilitate s release
Template strand positioned in active site
facilitated by channel in s
s3.2 loop blocks RNA elongation leading to
abortive transcription
Destabilization of -35 element s interactions
5 end of RNA
Promoter clearance and release of s
Rudder facilitates DNA unwinding during
elongation
Fr. Darst Curr Opin Struc Biol 2003
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The DNA lies across one face of the holoenzyme,
completely outside of the pol active site
channel All sequence-specific contacts
between pol and DNA are via the s subunit
RNA polymerase holoenzyme-promoter DNA structure
Transcription initiation
Fr. Murakami et al. Science 2961285 2002
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ß
Closed complex
Open complex
(? subunit shown semi-transparent)
Dk gr template DNA strand Lt gr nontemplate
strand Yellow - -10 -35 elements Red AT-rich
element, 1, and extended -10 region
Magnified view of RPo
Top view of RPo Stereo Image
Transcription Initiation Complexes
Fr. Murakami et al. Science 2961285 2002
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The transcription elongation complex (based on
Taq RNA pol structure)
Fr. Korzheva Mustaev Curr. Opin. Microbiol.
4119 2001
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Taq core RNA polymerase TEC
NTPs
Template strand red Non-template str.
yellow RNA transcript - gold ? subunit - cyan ?
subunit - pink
(NTP channel)
DNA protected from footprinting (gray)
Slicing Fig A above horizontally, and viewing
cross-section from top
Fr. Korzheva et al. Science 289619 2000
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Mechanism of hairpin-dependent pausing and
rho-independent transcription termination
Fr. Mooney et al. J. Bact. 1803265 1998
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Yeast RNA pol II
Movement of clamp (yellow) domain
DNA - purple RNA - green
Open/Free enz.
Closed/Elongating
Fr. Gnatt BBA 1577175 2002
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Yeast RNA Pol II
Fr. Cramer et al. Science 288640 2000
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Yeast Pol II-TFIIF complex
Model of Pol II-TFIIB-TBP-complex Pink
non-template strand Lt. blue template strand
Yellow TFIIB Green TBP
Blue pol II-TFIIF Red Pol II only
Fr. Chen Hahn Cell 2004
Fr.Chung et al. Molec Cell 2003
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(No Transcript)