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本帖最後由 非常時期 於 2010-10-4 01:29 編輯
RNA Polymerase II-associated Protein (RAP) 74 Binds Transcription
Shi Min Fang and Zachary F. Burton‡
From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
A set of deletion mutants of human RNA polymerase
II-associated protein (RAP) 30, the small subunit of transcription
factor IIF (TFIIF; RAP30/74), was constructed
to map functional domains. Mutants were tested for accurate
transcriptional activity, RAP74 binding, and
TFIIB binding. Transcription assays indicate the importance
of both N- and C-terminal sequences for RAP30
function. RAP74 binds to the N-terminal region of
RAP30 between amino acids 1 and 98. TFIIB binds to an
overlapping region of RAP30, localized to amino acids
1–176 (amino acids 27–152 comprise a minimal binding
region). The C-terminal region of RAP74 (amino acids
358–517) binds directly and independently to TFIIB. Interestingly,
RAP74 blocks TFIIB-RAP30 binding, both by
binding TFIIB and by binding RAP30. When the TFIIF
complex is intact, therefore, TFIIB-TFIIF contact is
maintained through RAP74. If the TFIIB-RAP30 interaction
is physiologically important, the TFIIF complex
must dissociate within some transcription complexes.
A minimal pathway for assembly of pre-initiation complexes
on RNA polymerase II-dependent promoters has been defined
in vitro (1–5). On promoters that include a TATA box, TBP1
(TATA-binding protein) first binds to this recognition sequence.
TFIIA facilitates this interaction. TFIIB can enter the complex
either by binding to these template-associated factors or by
binding to RNA polymerase II. For a tight association with the
complex, polymerase must first associate with TFIIF, so TFIIB
and TFIIF cooperate to bring polymerase into the pre-initiation
complex (4 –7). TFIIE and TFIIH are additionally required for
accurate initiation from linear DNA templates.
TFIIB is a single polypeptide of 33 kDa (8, 9) that can be
divided by sequence and mutational analysis into N- and Cterminal
regions. Near the N terminus is a probable Zn21-
binding sequence that is important for pre-initiation complex
assembly (10). Immediately adjacent to the Zn21-binding motif
is a highly conserved sequence that is important for selection of
transcriptional start sites (11). This N-terminal region of TFIIB
binds the RAP30 subunit of TFIIF and also RNA polymerase II
(12, 13), and mutants in this region inhibit assembly of transcription
intermediates, probably because they fail to interact
with either TFIIF or polymerase (10, 12–14). The C-terminal
region of TFIIB contains two imperfect direct repeats. Near the
end of the first repeat is a very basic sequence, and the region
surrounding this structure includes separate binding sites for
TBP and the acidic activation domain of herpes simplex virus
VP16 (15, 16). In the absence of other factors, the N- and
C-terminal regions of TFIIB fold on one another, masking
binding sites for other transcription factors. Binding to VP16
opens up TFIIB to expose the C-terminal region, which binds
TBP, and the N-terminal region, which binds RNA polymerase
II and TFIIF (16). Thus, TFIIB forms a bridge between TBP
and RNA polymerase II-TFIIF, in assembly of the pre-initiation
complex, and the assembly steps in which TFIIB is involved
are influenced by regulators. Recent reports of an x-ray
crystal structure of TATA box DNA with C-terminal fragments
of TBP and TFIIB (17) and a nuclear magnetic resonance
structure of a C-terminal fragment of TFIIB (18) support this
model for TFIIB function in assembly. The C-terminal region of
TFIIB binds TBP, activators, and template, and the N-terminal
region is presented as a scaffold for assembly of RNA polymerase
II-TFIIF into the complex (17, 18).
TFIIF is a heteromeric factor of 28- (RAP30) and 58-kDa
(RAP74) subunits (19–22), but there is some indication that in
certain contexts these subunits may enter complexes as separate
factors (7, 23, 24).
Accurate initiation has been demonstrated from highly supercoiled
templates using a system consisting of RNA polymerase
II, TFIIB, and either TBP (25) or YY1, which is an
initiator binding protein (26). One implication of these observations
is that RNA polymerase II and TFIIB might minimally
suffice to select transcriptional start sites. Consistent with this
view, swapping Schizosaccharomyces pombe for Saccharomyces
cerevisiae TFIIB and RNA polymerase II, in a system otherwise
comprised of S. cerevisiae factors, shifts the position of the
transcriptional start to that characteristic of S. pombe (27).
Also, some sua7 mutants in the S. cerevisiae gene encoding
TFIIB are altered for selection of initiation sites (28). sua8
mutants, in the gene encoding the largest subunit of RNA
polymerase II, affect transcriptional starts in a very similar
way to these sua7 mutants (29). Interestingly, mutations in the
S. cerevisiae SSU71/TFG1 gene, which encodes the homologue
of the RAP74 subunit of human TFIIF, suppress abnormal
start site selection in a sua7ssu71 double mutant (30). By itself
the ssu71 mutant does not affect transcriptional starts, so
TFIIF may suppress the sua7 mutant indirectly through another
general factor. The RAP30 subunit of human TFIIF has
been shown to interact physically with the N-terminal region of
TFIIB (12), so the large subunit of TFIIF interacts genetically,
and the small subunit interacts physically, with TFIIB.
The largest subunit of RNA polymerase II has an interesting
C-terminal domain (CTD) that consists of 52 repeats of the
consensus sequence YSPTSPS (31, 32). RNA polymerase II
* This work was initially supported by a grant from the National
Institutes of Health. This work was also supported by the Michigan
State University Agricultural Experiment Station, by Michigan State
University, and by Verna C. Finkelstein. The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked 「advertisement」 in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed. Tel.: 517-353-0859;
Fax: 517-353-9334; E-mail: burton@pilot.msu.edu.
1 The abbreviations used are: TBP, TATA-binding protein, RAP, RNA
polymerase II-associated protein; TF, transcription factor; CTD, Cterminal
domain of the large subunit of RNA polymerase II; ELISA,
enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis;
BSA, bovine serum albumin; PBS, phosphate-buffered
saline.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 20, Issue of May 17, pp. 11703–11709, 1996
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
11703
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發表於 2010-10-4 06:43
