RNA Polymerase II
with Transcription Factor IIB
Jason Cinti ('16) and Issam Hamdallah ('16)
Contents:
I. Introduction
In order for a gene to be
expressed, it must first be transcribed into mRNA, the precursor of all
proteins. The enzyme most responsible for the synthesis of mRNA is RNA
polymerase II (Pol II). By itself, Pol II is incapable of binding
upstream promoters and beginning the synthesis of mRNA. Instead, a
multitude of transcription factors (TFIIB, IID, IIE, and IIH) assemble
at the promoter DNA to recruit and properly place Pol II. Together, this
large complex of proteins, called the pre-initiation complex (PIC),
ensure accurate initiation because each contributing element must bind
in a specific order and in a precise arrangement before transcription
can occur.
Of the various components of the PIC, the TATA-box
binding protein (TBP) and transcription factor IIB (B) are responsible
for the successful delivery of Pol II to the promoter DNA as well as
promoter clearance. Specifically, B stabilizes TBP to the TATA-element
DNA to form the ternary complex, which in turn allows RNA polymerase II
to bind. Once RNA polymerase II binds to the ternary complex,
recruitment of the additional transcription factors to form the complete
PIC is made possible. Here we examine the structure of B and how B
interacts with various components of Pol II.
II. General Structure of
B:
The
polypeptide chain is composed of four key domains
.
At the N-terminus resides the B-ribbon.
The B-ribbon binds to the dock domain of Pol II and is composed of two
antiparallel beta sheets
and a pocket for a
zinc ion
.
A pair of cysteine residues (24
and 27, and 45 and 47) share a bond with the zinc ion at an average
distance of 0.243 nm. The next domain along the protein chain towards
the C-terminus is the B-reader
,
aptly named for its role in reading the DNA sequence during the
selection of a transcription start site (TSS). The
B-reader contains a single alpha helix that spans from residues 57-69
and loops to the B-linker
domain
.
The B-linker can be found in the
rudder and clamp coiled-coil domain of Pol II and is composed of one
beta strand (residues Asn 71 - Gly 86) and an alpha helix (residues
Thr 87 - Ser 110). Following the
B-linker is the B-core
,
which plays a key role in anchoring the TBP to the TATA-element DNA
and thus stabilizes the ternary complex. The B-core
is composed of an N-terminues cyclin region and a C-terminus cyclin
region connected by a small linker
region
.
Each cyclin fold is composed of five similar alpha helices, yet the
two folds differ because of the presence of a
short alpha helix on the
most outward region of the C-terminus fold
.
The B-core is able to anchor the
TBP to the TATA-box by interacting with both the DNA backbone and the
TBP. B-core binding
to the DNA backbone occurs non-specifically and is mediated by positively charged
residues that can interact with the negatively charged DNA
backbone both upstream and downstream of the TATA-box
.
The Coulombic attraction between the B-core and the DNA is crucial to
the stability of the TBP, TATA-box and B ternary complex. Further
stabilization of this complex can be attributed to the interaction
between the B-core and
TBP
.
It is a single loop
in the C-terminus of
TBP that interacts with the
positively charged residues of the B-core
.
Many of the residues form hydrogen bonds and Van der Waal interactions
with the B-core to stabilize the
complex, but the sulfide bond between residues
Lys-188 and Glu-146
particularly dominates the interaction
.
III. Interactions with
Pol II domains:
As soon as B secures TBP to the
TATA-box on DNA, RNA polymerase II can recognize the complex and bind.
The zinc ion in the B N-terminus creates a ribbon
like fold pocket - a structure very conducive to forming strong
interactions with other proteins. This pocket docks the B polypeptide
chain at a single beta strand in Pol II located in a small domain
called the dock domain
.
Disruptions in the zinc ribbon fold caused by mutations often result
in decreased levels of transcription, suggesting that the specific
folds created by the zinc ion facilitates easier recruitment of Pol
II. The B-ribbon residues Glu-40,
Leu-47, and Glu-26
are a few of many that help
stabilize the initial recruitment of Pol II by interacting with
dock domain residues Asp-414,
Ala-412, and
Asp-411,
respectively
.
B then threads through a region of Pol II called the RNA exit tunnel
and the B-reader makes contacts
with the active
center
.
The active center contains a magnesium ion and is the primary location
of messenger RNA catalysis. Proximate to the active center, the B-linker domain is found at the the
rudder and clamp
coiled coil domain
,
which is responsible for positioning the DNA during initiation and
maintaining the proper transcription bubble conformation. Finally, the
B-core
pushes up against the
hybrid binding / wall domain
,
which plays a key role in the elongation process by binding nascent
mRNA as it is generated. With Pol II secured to B
,
both of which are securlely anchored to TATA-element DNA, the
additional transcription factors can bind to form the PIC and
transcription can proceed.
V. References
(1) Bushnell D.A.,
Westover K. D., Davis R.E., Kornberg R.D. 2003. Structural Basis
of Transcription: An RNA Polymerase II?TFIIB Cocrystal at 4.5
Angstroms. Science 303: 983-988
(2) Liu X., Bushnell
D.A., Wang D., Guillermo C., Kornberg R.D. 2010. Structure
of an RNA Polymerase II-TFIIB Complex and Transcription
Initation Mechanism. Science 327: 206-209
(3) Hahn S., Roberts S.
2000. The zinc ribbon domains of the general transcription
factors TFIIB and Brf: conserved functional surfaces but
different roles in transcription initiation. Genes and
Development 14: 719-730
(4) Kostrewa D., Zeller M.
E., Armache K. J., Seizl M., Leike K., Thomm M., Cramer P.
2009. RNA polymerase II?TFIIB structure and mechanism of
transcription initiation. Nature 462: 323-330
(5) Nikolov, D.B., Chen, H., Halay, E.D., Usheva, A.A.,
Hisatke, K., Kun Lee, D., Roeder, R.G., Burley., S.K., Crystal
structure of a TFIIB-TBP-TATA-element ternary complex. Nature
377, 1190128
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