From the Department of Biochemistry and the Center for Advanced Molecular Biology and Immunology, School of Medicine and Biomedical Sciences, State University of New York, Buffalo New York 14214-3000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The general transcription factor IIB (TFIIB) is required for accurate and efficient transcription of protein-coding genes by RNA polymerase II (RNAPII). To define functional domains in the highly conserved N-terminal region of TFIIB, we have analyzed 14 site-directed substitution mutants of yeast TFIIB for their ability to support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and to form stable complexes with purified RNAPII in vitro. Mutations impairing the formation of stable TFIIB·RNAPII complexes mapped to the zinc ribbon fold, whereas mutations conferring downstream shifts in transcription start site selection were identified at multiple positions within a highly conserved homology block adjacent and C-terminal to the zinc ribbon. These results demonstrate that the N-terminal region of yeast TFIIB contains two separable and adjacent functional domains involved in stable RNAPII binding and transcription start site selection, suggesting that downstream shifts in transcription start site selection do not result from impairment of stable TFIIB·RNAPII binding. We discuss models for yeast start site selection in which TFIIB may affect the ability of preinitiation complexes to interact with downstream DNA or to affect start site recognition by a scanning polymerase.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Eukaryotic RNA polymerase II (RNAPII)1 requires the action of at least six accessory proteins to accurately initiate transcription. These accessory proteins, termed the general transcription factors (GTFs), have been the focus of much investigation and include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. The GTFs and RNAPII assemble in an ordered stepwise fashion on a class II promoter in vitro to form a functional preinitiation complex (PIC) (reviewed in Ref. 1). Assembly is initiated by the binding of TFIID to the TATA element via the TATA-binding protein (TBP) subunit, in some cases assisted by TFIIA. This complex is recognized by TFIIB, which binds and recruits RNAPII and TFIIF. PIC formation is completed by the association of TFIIE and then TFIIH, and the resulting complex can hydrolyze ATP and initiate mRNA synthesis. In contrast to this ordered-assembly model for PIC formation, it has been proposed that a preassembled holoenzyme, consisting of RNAPII, most of the GTFs, and additional factors, is recruited in one step to promoter-bound TFIID in vivo (reviewed in Ref. 1).
The general transcription factor TFIIB has an essential role in RNAPII transcription, and together with RNAPII and TBP, defines the minimal set of factors necessary for promoter-dependent transcription of a supercoiled DNA template in vitro (1). In both the ordered-assembly and holoenzyme-recruitment models of PIC formation, TFIIB recognizes promoter-bound TFIID and facilitates association of the remaining GTFs and RNAPII. Consistent with this role, TFIIB interacts with DNA adjacent to the TATA box (2) and binds to TBP (3-5), the TBP-associated factor TAF40 (6), RNAPII (4, 7, 8), and both subunits of TFIIF (4, 9). TFIIB may also play a role in the regulation of transcription by gene-specific regulatory proteins, as many of these regulatory factors bind TFIIB directly (10-17). In addition, TFIIB is involved in the selection of transcription start sites, as mutations in the Saccharomyces cerevisiae SUA7 gene, encoding TFIIB, can alter transcription start site selection in vivo and in vitro (7, 18).
The conservation of TFIIB structure among eukaryotic organisms
underscores its central role in transcription. The protein is comprised
of a highly conserved N-terminal region and a C-terminal core domain
(Fig. 1). Although no structural information has been obtained for
full-length TFIIB, NMR structures have been obtained for the core
domain of human TFIIB and for a portion of the N-terminal region of the
archaeal TFIIB from Pyrococcus furiosus (19, 20). In
addition, the crystal structure has been reported for the core domain,
as well as for a DNA·TBP·core TFIIB ternary complex (21). These
studies have revealed several structural motifs, including a zinc
ribbon fold in the N-terminal region, a pair of -helical direct
repeats in the core domain, and an amphipathic helix between the
repeats.
The high degree of amino acid conservation of the N-terminal region of TFIIB suggests that important functions reside in this portion of the protein. Reflecting this, random PCR mutagenesis of the yeast TFIIB gene identified a number of cold- and temperature-sensitive mutants that mapped to this region (22). To determine the functions of the N-terminal region and to define functional domains, we have undertaken a site-directed mutagenesis study of yeast TFIIB. In previous work, we identified two substitution mutants, L50D and R64E, that exhibited impaired RNAPII binding and a downstream shift in transcription start site selection, respectively (7). To extend our analysis and to define the functional domains involved in stable RNAPII binding and transcription start site selection, we have in this work analyzed 14 additional site-directed substitution mutants for their ability to support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and to bind purified RNAPII in vitro. Our data demonstrate that the N-terminal region of TFIIB contains two adjacent and separable functional domains involved in stable RNAPII binding and transcription start site selection.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids and TFIIB Site-directed Mutagenesis-- Yeast TFIIB substitution mutants were generated using the megaprimer method of PCR mutagenesis and plasmid p314/yIIBN as template as described previously (7). For mutants that exhibited altered properties in vivo, N-terminally polyhistidine-tagged derivatives expressed from the normal SUA7 promoter were generated by replacing the 1100-base pair NdeI-PstI fragment of plasmid p314/H6wt (containing the SUA7 coding region) with the corresponding fragment from the p314/yIIBN mutant plasmid as described (7). For the production of recombinant polyhistidine-tagged TFIIB proteins, the 1100-base pair NdeI-PstI fragment from the relevant p314/yIIBN mutant plasmid was isolated and inserted between the NdeI and PstI sites of bacterial expression plasmid pQE/yIIB (7).
In Vivo Analysis of TFIIB N-terminal Mutants--
To determine
the ability of the TFIIB mutants to support yeast cell growth, a
plasmid-shuffle complementation assay was used (7, 23). Test strain
FP153 (MAT, ura3-52, trp1
63,
sua7
1 and p316/yIIB[URA3]) contains a
deletion of the chromosomal sua7 coding region
(BclI-BamHI) with viability maintained by the
presence of a wild-type SUA7 gene on a URA3-based
vector. Strain FP153 was transformed with either wild-type or mutant
TFIIB constructs in plasmid p314 (TRP1 selectable marker),
selecting for Ura+, Trp+ colonies on CAA medium lacking uracil and
tryptophan at 30 °C (7). Transformants were grown in liquid CAA
medium lacking uracil and tryptophan at 30 °C, dilutions were
spotted on SC medium containing 5-fluoroorotic acid (5-FOA), and the
plates were incubated at room temperature for 4 days. 5-FOA, toxic to
cells containing a functional URA3 gene, selects for cells
that have spontaneously lost the URA3-containing plasmid
p316/yIIB with wild-type TFIIB. Thus, the appearance of 5-FOA-resistant
colonies reflects the ability of a TFIIB mutant to support cell
viability in the absence of wild-type TFIIB. To further analyze the
growth properties of mutants that supported viability, 5-FOA-resistant
colonies were grown to mid-exponential phase in liquid YPD (1% yeast
extract, 2% Bactopeptone, 2% dextrose) medium at room temperature,
and dilutions in sterile water were spotted on YPD plates. The plates were incubated at low (16 °C), intermediate (30 °C), or high
(37 °C) temperature (Table I). The mutant strains were also analyzed on a variety of other growth media, but the relative growth properties on these media were similar to those observed with YPD (data not shown).
Immunoblotting-- To determine the steady-state protein levels of TFIIB mutants with altered growth properties, strains containing both wild-type and polyhistidine-tagged mutant SUA7 alleles were grown to mid-exponential phase in liquid CAA lacking tryptophan and uracil. Cells were harvested by centrifugation and whole-cell extracts were prepared as described previously (24). Extracts (50 µg of protein) were resolved on a 10% SDS-polyacrylamide gel, transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore), and probed with a mouse monoclonal antibody raised against the polyhistidine-tag (RGS-His; Qiagen, 1:2,000 dilution) followed by goat anti-mouse IgG coupled to horseradish peroxidase (Jackson Laboratories, 1:40,000 dilution). Immune complexes were visualized by the addition of SuperSignal Ultra chemiluminescent substrate (Pierce) and autoradiography.
Purification of Recombinant Proteins-- Polyhistidine-tagged TFIIB proteins were expressed in Escherichia coli host strain JM109 under control of the phage T5 promoter in plasmid pQE32*. Culture growth, recombinant protein expression, and purification were carried out as described previously (7). Purified TFIIB proteins were judged to be >90% pure by SDS-PAGE.
In Vitro Transcription Reactions--
TFIIB-depleted whole-cell
extracts were prepared from strain FP133 as described (7). Strain FP133
(MAT, ura3-52, trp1, leu2, sua7-101,
his3::TRP1, prb1-1122, pep4-3, prc1-407, gal2) contains a temperature-sensitive allele of sua7 that results
from instability of the mutant TFIIB protein. At room temperature
(permissive), strain FP133 contains about 10% of the wild-type levels
of TFIIB protein, and the mutant TFIIB protein undergoes accelerated
turnover upon shifting to 37 °C. Transcription reactions contained
50 mM HEPES-KOH (pH 7.6), 10% glycerol, 50 mM
potassium acetate, 5 mM EGTA, 10 mM magnesium
acetate, 2.5 mM dithiothreitol, 2 units of Inhibit-ACE (5 Prime
3 Prime, Inc., Boulder, CO), 30 mM creatine phosphate, 1.5 units/ml creatine kinase, 333 µM
3'-o-methyl-GTP, 400 µM ATP, 400 µM CTP, 1.5 µM UTP, and 10 µCi of
[
-32P]UTP (3000 Ci/mmol). Supercoiled DNA template,
extract, and recombinant proteins were added as described in the figure
legends. Reactions were incubated at room temperature for 30 min,
stopped by the addition of 200 µl of stop buffer (10 mM
Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA and
10 units of RNase T1) and incubated for an additional 20 min.
Proteinase K (50 µg) and SDS (final concentration of 0.5%) were
added, and the resulting mixtures were incubated at 37 °C for 20 min. RNA transcripts were ethanol-precipitated and subjected to
electrophoresis on 7% denaturing polyacrylamide gels. Gels were dried,
and transcripts were visualized by autoradiography.
Mapping of in Vivo mRNA Start Sites-- Transcription start sites for the ADH1 and CYC1 genes were determined by primer extension analysis of total yeast RNA as described (25). The oligonucleotide primer for analysis of ADH1 mRNA contained the sequence 5'-dGTATTCCAACTTACCGTGGGATTCG-3', corresponding to positions +63 to +39 (where +1 is the initiating ATG). The oligonucleotide primer for analysis of CYC1 mRNA contained the sequence 5'-dGTCTTGAAAAGTGTAGCACC-3', corresponding to positions +53 to +34 (where +1 is the initiating ATG).
RNAPII Co-immunoprecipitation Assay-- Purified yeast RNAPII (500 ng) and TFIIB (100 ng) were incubated in 250 µl of P100 buffer (50 mM Tris acetate (pH 7.9), 10% glycerol, 100 mM potassium acetate, 14 mM magnesium acetate, 4 mM dithiothreitol, 1 mM EDTA, 50 µg/ml bovine serum albumin, and protease inhibitors (0.5 µg/ml pepstatin A, 0.5 µg/ml leupeptin, 2 µg/ml chymostatin, 1 µg/ml bestatin, 2.5 µg/ml antipain, 0.5 µg/ml aprotinin, 1 mM PMSF, 2 mM benzamidine)) at 4 °C overnight. Pansorbin (Calbiochem) was harvested by centrifugation and resuspended in blocking buffer (50 mM Tris acetate (pH 7.9), 100 mM potassium acetate, 25 mg/ml PVP-K-30 (U. S. Biochemical Corp.), 60 mg/ml casein, final solution brought to pH 7.9 with 1 M KOH). After incubation for 20 min at room temperature, the Pansorbin was harvested by centrifugation and resuspended to its original volume with P100 buffer, and 25 µl was added to each reaction. The samples were incubated for 30 min at 4 °C with gentle rocking and were centrifuged at 15,000 × g for 2 min. Supernatants were transferred to fresh tubes containing 2.5 µg of anti-RNAPII monoclonal antibody (8WG16, Research Diagnostics) and incubated for 90 min at 4 °C with gentle rocking. Pansorbin (pre-blocked, 25 µl in P100) was then added, and the reactions were incubated for 60 min at 4 °C with gentle rocking. The samples were centrifuged at 15,000 × g for 2 min, and the pellets were washed three times with 250 µl of buffer P100 containing 0.1% Nonidet P-40 and once with 250 µl of P100. The pellets were resuspended in 40 µl of 1× SDS sample buffer, and 10 µl of each sample was resolved on a 10% SDS-polyacrylamide gel and transferred to Immobilon-P polyvinylidene difluoride membranes. Immunoblotting was performed as described above, using an affinity-purified anti-yeast TFIIB antibody (1:5,000), followed by goat anti-rabbit IgG conjugated to horseradish peroxidase (Jackson Laboratories, 1:40,000).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of TFIIB N-terminal Substitution Mutants with
Altered Properties in Vivo--
The N-terminal region of TFIIB
contains several highly conserved sequence motifs (Fig.
1). To address the role of these motifs in the function of yeast TFIIB, we constructed and analyzed 14 site-directed substitution mutants. Substitutions were introduced at
conserved residues that reside within the second rubredoxin knuckle of
the zinc ribbon, the C-terminal -strand of the zinc ribbon, and an
adjacent homology block that is highly conserved among seven eukaryotic
species. The mutants were initially tested for their ability to support
yeast cell viability using a plasmid-shuffle complementation assay (see
"Experimental Procedures"). Plasmid-shuffling of the mutants
revealed that substitution mutants C45S/C48S and L52P did not support
cell growth (Table I). The remaining 12 mutants, capable of supporting viability, were tested for their growth
rates at several temperatures. Seven mutants were indistinguishable from the wild-type, whereas five mutants (W63P, W63R, R64A, F66D, and
H71E) exhibited varying degrees of cold sensitivity, with the W63P and
H71E mutants also displaying sensitivity to elevated temperature. The
mutants were also analyzed on a variety of other growth media, but the
relative growth properties on these media were similar to those
observed with YPD (data not shown).
|
|
|
In Vitro Transcriptional Analysis of TFIIB Mutants--
To
determine the transcriptional activity of the TFIIB mutants conferring
altered in vivo properties, we expressed and purified recombinant polyhistidine-tagged versions of the mutant TFIIB proteins
and tested their abilities to support basal and activated transcription
using a TFIIB-depleted whole-cell extract. The C45S/C48S double
substitution mutant did not support any detectable level of
transcription above background, whereas the L52P and W63P mutants were
severely compromised for transcriptional activity (Fig.
3). The H71E mutant was indistinguishable
from wild-type TFIIB, but significantly, the W63P, W63R, R64A, and F66D
mutant proteins all conferred preferential usage of the more downstream
transcription start site. The observed defects in transcriptional
activity of the mutant proteins are unlikely to be due to gross
misfolding, as all of the recombinant mutant proteins were competent to
form stable TBP·TFIIB·DNA ternary complexes in a gel mobility-shift assay (data not shown). In addition, all of the mutant proteins that
supported some basal transcription also supported a strong response to
the transcriptional activator GAL4-VP16 (Fig. 3). These results
demonstrate that the second rubredoxin knuckle and C-terminal
-strand of the zinc ribbon are critical motifs for basal
transcription activity, whereas the adjacent homology block participates in transcription start site selection.
|
Determination of Transcription Start Sites in Vivo-- The results of the in vitro transcription assays revealed that several of the mutants displayed an alteration in transcription start site selection on the CYC1 promoter. To confirm and extend these results, we examined in vivo start site utilization at the ADH1 and CYC1 promoters in the mutant strains. For comparison, we also analyzed RNA from strains containing the E62K and R64E substitution mutants, shown previously to alter start site utilization (7, 18). Consistent with the in vitro results, mutants W63P, R64A, and F66D displayed a downstream shift in start site selection from both the CYC1 and ADH1 promoters, whereas the shifts observed for mutant W63R were less pronounced (Fig. 4).
|
Analysis of RNAPII-TFIIB Interaction--
In previous work, we
identified a substitution mutant in the C-terminal -strand of the
zinc ribbon (L50D) that was unable to bind purified yeast RNAPII
in vitro (7). Although this result suggests that the zinc
ribbon is involved in the interaction between TFIIB and RNAPII,
additional mutants in the zinc ribbon would further establish its
participation in RNAPII binding. Therefore, we determined the ability
of the N-terminal mutants to form stable complexes with purified RNAPII
using a co-immunoprecipitation assay. The C45S/C48S and L52P mutants
did not form stable complexes with RNAPII, whereas the remaining
mutants did complex with RNAPII, with the W63P protein exhibiting only
slight impairment (Fig. 5). To confirm
participation of the N-terminal region of yeast TFIIB in stable RNAPII
binding, we tested the ability of recombinant core domain (residues
119-346) to form stable complexes with RNAPII. Full-length wild-type
or L50D TFIIB protein was included in the binding reactions to act as
internal positive and negative controls for stable RNAPII binding,
respectively. As shown in Fig. 6, the core domain of TFIIB did not detectably bind RNAPII in these assays. Taken together, these results demonstrate that the second rubredoxin knuckle and the C-terminal
-strand of the zinc ribbon in the N-terminal region of TFIIB are involved in the direct interaction between TFIIB and RNAPII.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this work, we have analyzed the in vivo and in
vitro properties of 14 site-directed substitution mutants in the
conserved N-terminal region of yeast TFIIB. Several structural motifs
were targeted for mutagenesis, including the second rubredoxin knuckle and C-terminal -strand of the zinc ribbon fold and an adjacent homology block that is conserved among seven eukaryotic species (Fig.
1). In vivo analyses revealed that two of the mutants,
C45S/C48S and L52P, were incapable of supporting yeast cell growth,
while an additional five mutants exhibited cold sensitivity and, in two
cases, temperature sensitivity as well (Table I). In vitro transcriptional analysis demonstrated that both the C45S/C48S and L52P
mutant proteins were severely impaired in their ability to support
basal transcription (Fig. 3). These transcriptional defects correlated
with an inability of these two mutant proteins to form stable complexes
with purified RNAPII in vitro (Fig. 5). Similarly, the core
domain, which lacks the N-terminal region and the zinc ribbon, also
failed to bind RNAPII (Fig. 6). These results, combined with our
previous determination that the L50D mutant is impaired for RNAPII
binding, strongly suggest that the zinc ribbon fold, and in particular
the C-terminal
-strand of the ribbon, are critical structural motifs
for stable interaction of TFIIB with RNAPII.
Four of the five mutants that displayed conditional growth properties contain a substitution in the homology block adjacent to the zinc ribbon (Fig. 1). The one conditional mutant with a substitution outside of this homology block, H71E, exhibited a temperature-sensitive phenotype but was comparable to the wild-type with respect to transcription activity in vitro, RNAPII binding, and transcription start site selection. The precise biochemical defect of the H71E mutant protein remains to be determined. The four conditional mutants with a substitution in the homology block (W63R, W63P, R64A, and F66D) exhibited cold-sensitive phenotypes and a downstream shift in transcription start site selection both in vivo and in vitro (Table I, Figs. 3 and 4). These results demonstrate that multiple residues within the highly conserved homology block adjacent to the zinc ribbon participate in selection of transcription start sites. In previous work, Hampsey and co-workers reported that E62K or R78C mutations conferred downstream shifts in start site selection (18). Residue Glu-62 is also contained within this homology block, whereas Arg-78 is C-terminal to this block, but also is an invariant residue among seven species (Fig. 1). In ongoing studies, we have utilized a random PCR-generated mutant TFIIB library and a genetic selection scheme to directly select for TFIIB mutants conferring downstream shifts in start site selection. To date, all of the mutants identified in this selection contain substitutions of residues in the homology block adjacent to the zinc ribbon or of residues Arg-78 or Val-79.2
Our results demonstrate that two separable and adjacent functional
domains in the N-terminal region of yeast TFIIB govern stable RNAPII
binding and transcription start site selection (Fig. 7). Mutations that impair stable RNAPII
binding, as determined by a co-immunoprecipitation assay, extend from
the second rubredoxin knuckle of the zinc ribbon through the C-terminal
-strand of the ribbon to residue Glu-62. We suggest that mutation of
the first rubredoxin knuckle of the zinc ribbon would also impair RNAPII binding, because it is highly likely that the integrity of the
ribbon fold is required for proper RNAPII interaction. Mutations
altering start site selection encompass residues Glu-62 to Phe-66, and
also include the more C-terminal pair of conserved residues Arg-78 and
Val-79. Mutants W63R, W63P, R64A, and F66D, all conferring downstream
shifts in start site selection, were proficient for stable RNAPII
binding. Moreover, the L52P mutant was defective for stable RNAPII
binding, but the low level of transcription supported by this mutant
protein initiated at normal start sites in vitro (Fig. 3).
Thus, our results indicate that downstream shifts in start site
selection are not caused by impairment in the stable association of
TFIIB and RNAPII. It was reported previously that the E62K and R78C
mutants, both conferring downstream shifts in start site selection,
were impaired for RNAPII binding in a surface plasmon resonance assay
(8). We suggest that residue Glu-62 is involved in both start site
selection and RNAPII binding, i.e. the adjacent domains for
stable RNAPII binding and start site selection overlap at residue
Glu-62.
|
We have determined that substitutions of residue Arg-78 other than R78C, such as R78L, confer downstream shifts in start site selection without impairing stable RNAPII binding by our assay (7).2 The failure of the R78C mutant to bind RNAPII perhaps results from the introduction of a cysteine residue adjacent to the zinc ribbon. The presence of this additional cysteine could interfere with normal coordination of zinc by the adjacent four cysteines in the zinc ribbon, thereby perturbing the ribbon structure and impairing RNAPII binding.
The mechanism by which transcription start sites are selected by RNAPII remains to be determined. In higher eukaryotes, transcription initiation usually occurs at a discrete start site located 25-30 base pairs downstream of the TATA element. In contrast, transcription initiation in S. cerevisiae frequently occurs at multiple start sites within a window of 30-120 base pairs downstream of the TATA element (26, 27). The window for transcription initiation in S. cerevisiae could result from preinitiation complexes that loop out a limited amount of intervening downstream DNA to interact with potential initiation sites. If so, TFIIB mutants could confer downstream shifts in start site selection by altering preinitiation complex structure so as to impair the ability to loop out shorter segments of DNA, thereby impairing the ability of the complexes to utilize start sites closer to the TATA element. However, it has been reported that promoter melting for the S. cerevisiae GAL1 and GAL10 genes in vivo is comparable to other eukaryotes, occurring approximately 20 base pairs downstream of the TATA element (28). The extent of promoter melting appears to be independent of the distance between the TATA box and the transcription start sites, suggesting that the positioning and subsequent promoter melting of S. cerevisiae preinitiation complexes is similar to mammalian preinitiation complexes. Thus, transcription initiation in the downstream window from S. cerevisiae TATA elements may be the result of a "scanning" polymerase (28). If start site selection in S. cerevisiae involves a scanning polymerase, upstream or downstream shifts in start site utilization could result from RNAPII with enhanced or diminished initiation site recognition, respectively. Upstream shifts in start site selection have been observed both in vitro and in vivo with yeast strains containing a deletion of the small RPB9 subunit of RNAPII or mutation of the C-terminal zinc-binding motif in RPB9 (29-31). Mutation or deletion of the RPB9 subunit might alter the structure of polymerase on the DNA such that initiation sites are more efficiently recognized early in the scanning process. Conversely, downstream shifts, conferred by mutations in TFIIB or the RPB1 subunit of RNAPII (32), may be a consequence of a polymerase with diminished start site recognition. TFIIB might function in start site selection as a component of the scanning polymerase complex and assist in initiation site recognition and/or utilization (18). If so, mutations that impair the stable association of TFIIB and RNAPII might produce unstable scanning complexes, resulting in alterations in start site selection.
Although it remains possible that TFIIB is part of a scanning polymerase complex, our results demonstrate that most mutations that confer downstream shifts in start site selection do not affect stable association of TFIIB and RNAPII. Moreover, the L50D and L52P mutants, defective for stable RNAPII binding yet able to support low levels of transcription, support initiation at normal start sites. Therefore, if a scanning polymerase is involved in start site selection, our results are more consistent with TFIIB conferring properties for initiation site recognition upon RNAPII at the time of preinitiation complex assembly. In this model, the zinc ribbon of TFIIB has the major function in the stable interaction with RNAPII, whereas the adjacent homology block may interact with RNAPII in a more subtle manner to affect start site selection. Such an interaction between the homology block of TFIIB and RNAPII could affect the conformation of the polymerase on the DNA, thereby affecting subsequent start site recognition upon the onset of scanning. Continued biochemical analyses of the TFIIB mutants described here should provide additional insight into the mechanism of transcription start site selection and the role played by TFIIB.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Lynne Pajak for technical assistance and Leo Faitar, V. James Hernandez, and Joe Napoli for helpful discussions and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Public Health Service Grant GM51124 from the National Institutes of Health (to A. S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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.: 716-829-2473;
Fax: 716-829-2725; E-mail: asp{at}acsu.buffalo.edu.
1 The abbreviations used are: RNAPII, RNA polymerase II; TF, transcription factor; GTF, general transcription factor; PIC, preinitiation complex; TBP, TATA-binding protein; PCR, polymerase chain reaction; 5-FOA, 5-fluoroorotic acid; PAGE, polyacrylamide gel electrophoresis; CAA, casamino acid.
2 S. L. Faitar and A. S. Ponticelli, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|