J Biol Chem, Vol. 274, Issue 40, 28736-28744, October 1, 1999
Alignment of the B" Subunit of RNA Polymerase III Transcription
Factor IIIB in Its Promoter Complex*
Sheila M. A.
Shah
,
Ashok
Kumar,
E. Peter
Geiduschek, and
George A.
Kassavetis§
From the Department of Biology and Center for Molecular Genetics,
University of California, San Diego,
La Jolla, California 92093-0634
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ABSTRACT |
TFIIIB, the central transcription initiation
factor of the eukaryotic nuclear RNA polymerase (pol) III is composed
of three subunits: the TATA-binding protein; Brf, the TFIIB-related
subunit; and B", the Saccharomyces cerevisiae,
TFC5 gene product. The orientation of the B" subunit within
the TFIIIB-DNA complex has been analyzed at two promoters by two
approaches that involve site-specific photochemical protein-DNA
cross-linking: a collection of B" internal and external deletion
proteins has been surveyed for those deletions that alter the
interaction of B" with DNA or change the orientation of B" relative to
DNA; a method for regionally mapping cross-links between specific DNA
sites and 32P-end-labeled protein has also been applied.
The results map an N-proximal segment of B" to the upstream end of the
TFIIIB-DNA complex and amino acids 299-315 to the principal
DNA-contact site, approximately 8 base pairs upstream of the TATA box.
The analysis also indicates that a segment comprising amino acids
316-434 loops away from DNA, and locates the C-proximal 170 amino
acids of B" downstream of the TATA box. Examination of two-cross-link
products formed by DNA with adjacent and nearby photoactive nucleotides supports the conclusion that Brf and B" share an extended interface along the length of the TFIIIB-DNA complex.
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INTRODUCTION |
The eukaryotic (nuclear) RNA polymerases are brought to their
promoters by relatively complex core transcription apparati. In the RNA
polymerase (pol)1 III
transcription system of Saccharomyces cerevisiae, which is the focus of this work, the polymerase recruitment function is executed
by the core transcription factor (TF) IIIB. TFIIIC and TFIIIA, the
other components of the core transcription apparatus, bind DNA and
serve as assembly factors for TFIIIB; the six-subunit TFIIIC interacts
directly with TFIIIB, and TFIIIA serves as a 5 S rRNA
gene-specific platform for TFIIIC. TFIIIB is composed of three
subunits: the TATA-binding protein (TBP), Brf, and B" (Tfc5). TBP
co-directs binding of TFIIIB to very strong TATA boxes. At promoters
that lack these intrinsic TBP-binding sites, TFIIIC functions as an
assembly factor that deposits TFIIIB on its upstream DNA site (reviewed
in Ref. 1). In both kinds of situations, TBP is located close to DNA
(2).
The 596-amino acid Brf is joined from two evolutionarily distinct
parts. Its designation as the TFIIB-related
factor derives from the homology of its N-proximal half to
TFIIB (3-5). Just as TFIIB is able to recruit pol II to the
transcriptional start site, so the principal polymerase recruitment
capacity of TFIIIB resides in the corresponding, N-proximal, half of
Brf. The C-proximal half of Brf is pol III-specific (6), and has no
sequence-homologous counterparts in the pol I and pol II transcription
apparati. The principal TBP and B" affinities of Brf, and also its
TFIIIC affinity, reside in this C-proximal half (7). However, the
N-proximal half of Brf alone has sufficient affinity for TBP and B" to
form a TFIIIB-DNA complex at a strong TATA box that is able to recruit pol III to accurately initiated transcription (7).
Since TFIIB and the N-proximal half of Brf sit in corresponding
locations in their respective DNA complexes (7, 8), and exercise
similar functions, it is surprising that Brf and TBP are not by
themselves competent to direct transcriptional initiation by pol III.
In fact, the 594-amino acid B" is absolutely required for transcription
by pol III, in duplex DNA or chromatin, at TATA-containing and
TATA-less promoters, in vivo as well as in vitro.
It is also B" that makes the TFIIIB-DNA complex extraordinarily stable
(9).
In the experiments that are reported here, we have mapped B" along its
DNA site in the TFIIIB-DNA complex; B" external and internal deletion
proteins that form stable TFIIIB-DNA complexes have been examined by
photochemical protein-DNA cross-linking along the extended DNA site in
order to find out which of these deletions alter the interaction of B"
with DNA or change the orientation of B" relative to DNA. A relatively
simple and highly sensitive method has also been developed to map
cross-links from specific DNA sites to their targeted region on
32P-end-labeled protein.
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MATERIALS AND METHODS |
Synthesis of DNA Probes for Photochemical Cross-linking--
The
SUP4 tRNA gene was polymerase chain reaction-amplified from
plasmid pTZ1 (10) with primers creating a BspE1 site beginning at bp
64 (+1 designating the start site of transcription) and an NsiI site
beginning at bp +124. The DNA was purified on native 5% polyacrylamide
gel, passively eluted (11), restricted with BspEI and
NsiI, and 5' end-labeled with T4 polynucleotide kinase. The
191-nucleotide non-transcribed and 183-nucleotide transcribed strands
were separated on 5% polyacrylamide, 8 M urea gel,
excised, and recovered by passive elution (11). The transcribed
strand was used to construct DNA with site-specifically placed
photoactive nucleotides, as described (12). Cross-linking probes were
synthesized by annealing specific oligonucleotides to the transcribed
strand. Primer extension to add ABdUMP
(5-[N-(p-azidobenzoyl)-3-aminoallyl]-deoxyuridine monophosphate) and [32P]dNMP utilized 1 unit of
exonuclease-free Klenow fragment (incubation for 5 min at 37 °C with
10 µM ABdUTP and 0.5 µM appropriate
[
-32P]dNTP). Extension of the photoactive DNA strand
was completed by an additional 10-min incubation with 500 µM unlabeled dNTPs. Probes were generated with ABdUMP at
bp 38/37,-33/32, 30, 26, 22/21, 19/17, 14/12, and
3/2 of the non-transcribed strand (Fig. 1A). For probes
19/17, 14/12, and 3/2, this process left an upstream,
22-nucleotide 3' overhang. An upstream oligonucleotide (the same as the
primer for making the 38/37 probe) was annealed and ligated to
these constructs as the final step in preparing the corresponding
photoactive 183-bp DNA.
SNR6 photoprobes 39/38, 33, 28, 13/12, and 5
were made as just described, using the appropriate non-transcribed
strand oligonucleotides annealed to an 88-mer transcribed strand
spanning bp 56 to bp +32. SNR6 photoprobes 42 and
23/22 were made using the appropriate non-transcribed strand
oligonucleotide primer annealed to a 60-mer transcribed strand template
spanning bp 58 to bp +2.
Proteins--
Expression plasmids for B"-(1-370) and
B"-(371-594) were constructed as described and referenced for other B"
deletions (11). The corresponding proteins were overproduced in
Escherichia coli BL21(DE3), (11). TFIIIC, wild-type TBP,
TBPm3, Brf, Brf-(1-282), Brf-(284-596), truncated and full-length B"
were purified as described (7, 13-15). Concentrations of TBP,
full-length B" and B"-(138-594) were determined by their predicted
extinction coefficients. Concentrations of TBPm3, Brf, and other B"
deletion proteins were estimated by Coomassie stain on
SDS-polyacrylamide gels, standardized to bovine serum albumin. TFIIIC
concentration was measured as described (9).
[32P]B"-(138-594)--
Ten pmol of B"-(138-594)
was incubated for 30 min at 21 °C with 25 units of bovine heart
muscle kinase and 5 µM [
-32P]ATP (6,000 Ci/mmol) in buffer containing 20 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 100 mM NaCl, and 2.5 mM dithiothreitol (11) for 32P labeling of its
natural phosphorylation site at Ser-164. Unincorporated [-32P]ATP was removed, and efficiency of
phosphorylation determined as described (11).
Photoaffinity Labeling of Proteins--
Protein-SUP4
gene complexes were allowed to form for 60 min at 21 °C in 20 µl
of pol III buffer (40 mM Tris-Cl (pH 8.0), 100 mM NaCl, 7 mM MgCl2, 3 mM 
-mercaptoethanol, 4-6% (v/v) glycerol, and 100 µg/ml bovine serum albumin) containing 0.2-2 fmol of SUP4 photoprobe, 18.7 fmol of TFIIIC, 200 fmol of B" (full-length or mutant), 50 fmol of TBP, 64 fmol of Brf, and 200 ng of pLNG56 (nonspecific carrier) DNA (9). After incubation with heparin (200 µg/ml), samples were UV-irradiated for 5 min, as described (16). A
6-µl aliquot of each sample was loaded on non-denaturing 4%
polyacrylamide gel containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, and 4% (v/v) glycerol (with 20 mM
Tris, 2 mM EDTA running buffer). The remaining material of
each sample was treated with DNase I (7.5 units) and S1 nuclease (80 units), and resolved by 8% SDS-PAGE. 32P-Tagged
(cross-linked) proteins were visualized and quantified by
phosphoimaging using software provided with the imager. TFIIIB complexes were assembled on SNR6 photoprobes with 400 fmol
of TBPm3, 64 fmol of Brf, 200 fmol of B" (full-length or mutant), and 5 fmol of an SNR6 photoprobe, incubated for 60 min at 21 °C in 20 µl of pol III buffer containing 50-70 mM (instead
of 100 mM) NaCl and 100 ng of poly(dG-dC)·poly(dG-dC)
(instead of pLNG56). Reaction mixtures were treated with 200 ng of
poly(dA-dT)·poly(dA-dT) (in place of heparin), UV-irradiated, and
processed as described above and elsewhere (12).
Partial Cleavage of Cross-linked [32P]B"-(138-594)
with CNBr and 2-Nitro-5-thiocyanobenzoic acid
(NTCB)--
Photochemically cross-linked reaction mixtures were
prepared with 800 fmol of TBPm3, 64 fmol of Brf, 80 fmol of
32P-labeled B"-(138-594), and 50-100 fmol of the
appropriate unlabeled SNR6 photoprobe in 20 µl of pol III
buffer (with 40-50 mM in place of 100 mM
NaCl). For protein complexes with the SUP4 gene, 30 fmol of
the appropriate unlabeled photoprobe was incubated with 18.7 fmol of
TFIIIC, 100 fmol of TBP, 64 fmol of Brf, and 33 fmol of
32P-labeled B"-(138-594) in 20 µl of pol III buffer
(with 100 mM NaCl). After UV irradiation, 0.6 pmol of
unlabeled B" was added (to reduce the background due to free
32P-labeled B") and proteins were resolved by 6% SDS-PAGE
to separate DNA-cross-linked 32P-labeled B" from free B".
The corresponding bands were visualized by phosphoimaging and excised
from the gel. Proteins were passively eluted overnight into buffer
containing 10 mM Tris-Cl (pH 8.0), 0.2% (w/v) SDS, 0.1 mM dithiothreitol, and 25 µg/ml bovine serum albumin, and
concentrated by ultrafiltration.
Eluted proteins were subjected to partial cleavage with 100 mM CNBr or 10 mM NTCB. For CNBr partial
cleavage, samples were brought to low pH with 1% SDS and 0.05 N HCl. CNBr (100 mM) was then added for 20 min
at 21 °C. Further reaction was quenched by adding 0.25 volumes of
100 mg/ml dithiothreitol in 1 M NaHepes (pH 7.8). For NTCB
partial cleavage, samples were treated with 10 mM NTCB for
30 min at 37 °C for cyanylation of cysteine residues. The pH was
then adjusted to 9.0 with Tris base, and samples were incubated
overnight at 37 °C for the subsequent proteolytic cleavage. The
32P-labeled fragments generated by NTCB or CNBr cleavage
were resolved on 11-15% SDS-polyacrylamide gels. Comparisons of
cleavage patterns of free and DNA-cross-linked 32P-labeled
B" were made on phosphoimage profiles.
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RESULTS |
Photochemical Cross-linking of B" Deletion Mutants--
Previous
examination of the internal structure of a TFIIIB-DNA complex by
photochemical cross-linking revealed that B" cross-links to DNA between
43 and 2 bp upstream of the transcriptional start site (16, 17). In
order to map the locations of individual domains of B" relative to DNA,
we have analyzed the photochemical cross-linking patterns of B" with
internal and external deletions, using ABdUMP incorporated at specific
sites along the TFIIIB-binding sites of the SUP4 and
SNR6 genes (Fig. 1). Each DNA
photoprobe also has a radioactive nucleotide incorporated next to, or
in close vicinity to, its photoactive nucleotide(s). TFIIIB complexes containing either full-length or truncated B" were assembled on each of
these DNA photoprobes; reaction mixtures were then UV-irradiated, digested with nucleases, and analyzed by SDS-PAGE. The efficiency of
TFIIIB-DNA complex formation was monitored in parallel by
electrophoretic mobility-shift analysis of an UV-irradiated, but not
nuclease-treated, portion of each reaction mixture. If deletion of a
specific segment of B" results in the loss of cross-linking at a
particular DNA site, this suggests localization of that peptide segment
in the vicinity of that DNA site (so long as complex formation is not correspondingly diminished). In the case of the SNR6 gene
promoter, unidirectional assembly of TFIIIB-DNA complexes is specified
by a modified TATA-box (TGTAAATA) in conjunction with the mutant TBPm3
(15). At the SUP4 tRNA promoter, with its very weak TATA box, TFIIIB-DNA complex assembly requires the transcription factor TFIIIC, and is unidirectional (15).
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Fig. 1.
Placement of photoactive nucleotides in the
TFIIIB-binding region of the SUP4 gene
(A) and the modified SNR6 gene
(B) for cross-linking. Locations of the
photoactive nucleotide, ABdUMP, are indicated with U.
Photoprobes are referred to by the position(s) of the ABdUMP relative
to the transcription start site. All U-for-T substitutions in the
SUP4 gene are in the non-transcribed strands. U-for-T
substitutions in the SNR6 gene transcribed strand are
indicated by a. The DNase I footprints of TFIIIC and TFIIIB
are indicated by striped and shaded boxes, respectively, below the DNA sequences.
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The SUP4 Promoter--
Efficiencies of forming heparin-stable
TFIIIB-DNA complexes and photochemical cross-linking of each B"
deletion mutant and wild type B" were compared for the photoactive DNA
probes shown in Fig. 1A. B" lacking its N-terminal 262 amino
acids or its C-terminal 130 amino acids remains competent to assemble
into a heparin-resistant TFIIIB-DNA complex via the
TFIIIC-dependent assembly pathway on the SUP4
gene (but does so relatively inefficiently; Ref. 11). Full-length B"
cross-linked most efficiently to ABdUMP positioned between bp 38 and
bp 32 on the SUP4 gene (Fig.
2A; cf. Ref. 16).
Removal of 223 or 262 amino acids from the N terminus of B" increased
the efficiency of cross-linking between bp 38 and bp 30 more than
2-fold compared with full-length B" (adjusted for differences in
formation of heparin-resistant complexes; Table I, part A, and Fig. 2A).
Further downstream, between bp 26 and 2, the cross-linking
efficiencies of B"-(224-594) and full-length B" did not differ
significantly.
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Fig. 2.
Cross-linking of B" deletion proteins to the
SUP4 and SNR6 genes. Photoprobes
were incubated with TFIIIC (SUP4 gene only), TBP, Brf, and:
A, full-length B" or B"-(224-594); B,
full-length B" or B"-(40-487); C, full-length B" or
B" 291-310; or D, full-length B" or internal deletion
variants of B". After UV irradiation, reaction mixtures were
nuclease-treated and 32P-tagged proteins were separated by
SDS-polyacrylamide gel electrophoresis. Photoprobes are identified
below each lane of panels A and
B, and the cross-linked proteins are identified at either
side of the panels. A, comparison of the cross-linking
profiles of intact B" and B"-(224-594) along the SUP4 gene.
B, comparison of the cross-linking profiles of intact B" and
B"-(40-487) along the SUP4 gene. C,
incorporation of B" and B"291-310 into the CB' complex
differentially affects cross-linking of the 120 kDa subunit of TFIIIC
(24). D, effects of B" internal deletions on cross-linking
to bp 39/38 of the SNR6 gene.
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Table I
B" deletions: effects on patterns of cross-linking to the SUP4 and SRN6
genes
++, >2× cross-linking relative to full-length B"; +, 0.5-2×
full-length B"; +/, 0.1-0.4× full-length B"; , <0.1×
full-length B"; ND, not done.
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The combination of low level formation of heparin-resistant TFIIIB-DNA
complexes with B"-(263-594) and low efficiency cross-linking of B"
between bp 26 and bp 2, resulted in cross-linking signals too close
to background to be reliably quantified, but the observed levels were
consistent with unimpaired cross-linking of B"-(263-594) at these
positions (Table I, part A). C-terminally truncated B"-(1-464) and
B"-(1-487) were not sufficiently well resolved from Brf in SDS-PAGE to
quantify reliably. It was, however, possible to assess that B"-(1-487)
was not significantly impaired in cross-linking to bp 38/37,
33/32, and 30, where B" cross-links efficiently (Table I, part
A). N-and-C-terminally truncated B"-(40-487) did resolve well from
Brf, and its cross-linking to all probes tested was unimpaired (Table
I, part A, and Fig. 2B). These results indicate that the
cross-links of B" to the SUP4 gene (more specifically to the
bp 38/2 segment of this gene) primarily involve its amino acid
224-487 segment; this is also the active core of B" for
SUP4 transcription (11). Internal deletion mutants of B",
which span much of the region of B" not covered by the N- and
C-terminal truncations, and which are capable of being assembled into
heparin-resistant DNA complexes, were also examined. Only B" with amino
acids 291-310 deleted displayed a defect in cross-linking,
predominantly with probe 38/37 for which cross-linking efficiency
is reduced more than 10-fold (Fig. 2C, lanes
3 and 5) and, to a lesser extent, with probes
33/32 and 30 (Table I, part A).
Fig. 2C shows two other aspects of this defect. 1) Heparin
did not diminish cross-linking of intact B" (lanes
2 and 3), but substantially reduced cross-linking
of B"291-310 without substantially reducing the cross-linking of
Brf (lanes 4 and 5). 2) Full-length B"
displaced the 120-kDa subunit of TFIIIC from the vicinity of the
photoactive nucleotide in the 39/38 photoprobe (compare lanes 1 and 2), but B"291-310 did
not (lane 4).
The SNR6 Promoter--
On the SNR6 gene (18, 19), B"
truncated by N-terminal deletion to amino acid 186 or by C-terminal
deletion to amino acid 487 was not deficient in cross-linking (Table I,
part B). N-terminal truncation to amino acid 224 or 263 somewhat
lowered cross-linking at bp 13/12 and 5 (20-40% of that
obtained with intact B"; Table I, part B), but a comparable depression
of cross-linking was not observed when TFIIIB complexes with the
SUP4 gene were probed at the close-by 14/12 and 3/2
sites (Table I, part A). Since no single region of B" is required for
TFIIIB-SNR6 DNA complex formation, a more complete set of
internal deletions, spanning regions not covered by N- and C-terminal
truncations, could be examined at this promoter. B" cross-links most
efficiently to bp 39/38 on the SNR6 gene (7); only at bp
33 and 13/12 do cross-linking efficiencies exceed 10% of
cross-linking to bp 39/38. Only B"272-292 was deficient in
cross-linking to probe 39/38 (20% of the efficiency of
cross-linking to intact B"; Table I, part B, and Fig. 2D).
Curiously, B"291-310, which was deficient for cross-linking at a
similar location on the SUP4 gene, was not significantly
impaired for cross-linking on the SNR6 gene (Table I and
Fig. 2; B"272-292 was not examined on the SUP4 gene
because it does not assemble into a stable, heparin-resistant complex;
Ref. 11). B"424-438 displayed somewhat lower cross-linking to bp
28 and 13/12 (20-40% of the cross-linking efficiency with
intact B") and B"409-421 was somewhat impaired in cross-linking to
bp 5 (40% efficiency relative to intact B").
Comparisons of cross-linking to the SUP4 and SNR6
genes are complicated by the fact that TFIIIC positions TFIIIB somewhat heterogeneously onto the AT-rich upstream sequence of the
SUP4 gene (20), whereas assembly on the SNR6 gene
directed by TBPm3 is fixed at the TGTA box. One would therefore expect
a "blurring" in the B" cross-linking pattern to the SUP4
gene relative to SNR6 (i.e. efficient
cross-linking between bp 38 and 30 on the SUP4 gene, but
predominantly at bp 39/38 on the SNR6 gene). Conversely, even if as few as 2% of TFIIIB complexes on the SNR6 gene
were bound in the reverse orientation (cf. Ref. 15), they
might make a significant contribution to cross-linking at bp 13/12
(through the B" segment that cross-links efficiently to bp 39/38 in
the opposite orientation of TFIIIB).
Split B"--
B" assembly into a TFIIIB-DNA complex buries two
regions that are more surface-exposed in the free protein (as judged by
hydroxyl radical footprinting): region I, covering amino acids
~390-470; and region II, covering amino acids ~270-305 (11). The
above experiments clearly suggest that region II lies close to the
major sites of B" cross-linking on the SNR6 and
SUP4 genes (since B"291-310 depressed cross-linking to
the SUP4 gene and B"272-292 to the SNR6
gene). In order to gain further information about the location of
region II relative to DNA, B" was split at amino acid 370 (which is
highly accessible to hydroxyl radical cleavage in TFIIIB-DNA complexes;
Ref. 11). Heparin-resistant TFIIIB-DNA complexes can be formed on both
the SUP4 and SNR6 genes only when both parts of
this split B" (amino acids 1-370 and 371-594) are provided together.
The transcriptional activity of these TFIIIB-DNA complexes is, however,
diminished (data not shown). Fig. 3
compares the cross-linking profiles of wild type and split B" on the
SUP4 and SNR6 genes. Cross-linking to the
SUP4 gene at bp 38/37, 33/32, and 30 is
contributed almost entirely by amino acids 1-370 of B", but there is
some cross-linking of the C-terminal amino acids 371-594 to bp
33/32 (Fig. 3A). The weak cross-linking of intact B" to
sites downstream of bp 30 on the SUP4 gene appears to
involve solely its C-terminal half. Similarly, B"-(1-370) dominates
cross-linking at bp 39/38 of the SNR6 gene (Fig.
3B), but some cross-linking of the C-terminal segment is
also apparent at this site. In contrast to the cross-linking pattern at
bp 33/32 of the SUP4 promoter, most of the cross-linking
of B" at bp 33 of SNR6 is contributed by its C-terminal
segment. As on the SUP4 gene, most of the C-terminal B"
segment cross-links also to bp 28, 13/12, and 5, but low level
cross-linking of the N-terminal segment at these sites also persists.
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Fig. 3.
N- and C-terminal segments of B" primarily
cross-link at the upstream and downstream ends, respectively, of its
DNA site. TFIIIB complexes containing either full-length B" or
both B"-(1-370) and B"-(371-594) were assembled on the
SUP4 (A) and SNR6 (B)
photoprobes that are identified above each lane. Cross-linked proteins
are indicated at both sides of the panels.
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A High Molecular Weight Photoadduct--
TFIIIB-DNA complexes with
the 38/37 SUP4 probe, which contains two ABdUMP
residues, yielded a high molecular weight material cross-linking
adduct, whose mobility depended on the B" external deletion mutant used
(Fig. 4, lanes 1 and 3). Such high molecular weight bands have been
characterized as the products of multiple cross-linking events (21).
That this particular multiply cross-linked product contains both B" and
Brf was demonstrated in the following way. The mobility of the high
molecular weight band increased when B" was truncated, indicating that
this DNA-protein adduct contains B" (Fig. 4, lane
3). That it also contains Brf was shown by using split Brf.
The two Brf fragments, Brf-(1-282) and Brf-(284-596), together form a
stable and transcriptionally fully competent TFIIIB complex on the
SUP4 gene (7). The cross-linked products of the
TFIIIB-SUP4 gene complex formed with intact and split Brf also yielded high molecular weight bands with different electrophoretic mobilities (Fig. 4, lane 2). Since the high
molecular weight Brf-B"-DNA photochemical adduct has also been observed
in UV-irradiated TFIIIB complexes formed on SUP4 photoprobes
33/32, 22/21, 19/17, 14/12, and 3/2 (data not
shown), we conclude that Brf and B" share an extended interface with
DNA.
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Fig. 4.
Multiply cross-linked protein-DNA adducts
show that both Brf and B" are bound in vicinity to bp 38 and 37 of
the SUP4 gene. Photoprobe 38/37 was incubated
with TFIIIC, TBP, full-length Brf or split Brf ((Brf-(1-282) and
Brf-(284-596)), and full-length B" or B"-(224-594), as indicated
above the lanes. Cross-linked polypeptides are identified at either
side of the panel.
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Partial CNBr Cleavage of B"-DNA Photoadducts--
Use of internal
deletions to map the orientation of a protein along its DNA site is
subject to the restriction that those deletions should not drastically
change the protein-DNA alignment. A second approach, which is free of
that restriction, was developed for mapping specific B" segments to the
vicinity of specific sites on the SUP4 and SNR6
genes. An example of the mapping procedure is shown in Fig.
5. N-proximally 32P-labeled
B"-(138-594) was used to form TFIIIB-DNA complexes with unlabeled
39/38 SNR6 photoprobe and subjected to UV-irradiation (Fig. 5A). The [32P]B"-DNA adduct was
recovered from a 6% polyacrylamide-SDS gel (Fig. 5B,
lane 3) and subjected to partial CNBr cleavage.
The cleavage products were then resolved on a 13% polyacrylamide-SDS gel (Fig. 5C). The partial cleavage patterns of free
B"-(138-594) is shown in lane 2 of
panel C, with the methionine C-terminal to each
cleavage site identified at the left of the panel. The partial digestion pattern of the B"-(138-594)-DNA adduct and free B"-(138-594) were identical to Met-298, but products of cleavage at
Met-315, -372, -379, and -425/434 were shifted to lower mobility due to
their attached DNA (lane 3). This specifies that
the major site of cross-linking of B" to the SNR6 gene at bp
39/38 is situated between amino acids 299 and 315. An identical
site between Met-298 and Met-315 was identified for the multiply
cross-linked products identified in Fig. 5B (data not
shown).
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Fig. 5.
A method to identify the polypeptide segment
located in the vicinity of a specific DNA site. A, the
scheme for partial CNBr cleavage of [32P]B"-(138-594)
cross-linked to the SNR6 gene at bp 39 and 38.
B, unlabeled SNR6 photoprobe 39/38 was
incubated with [32P]B"-(138-594), Brf, and TBPm3, as
indicated above the lanes. The UV-irradiated TFIIIB-DNA complexes were
denatured and resolved by SDS-PAGE. A phosphoimage of the wet gel is
shown. Bands of DNA-linked and free B"-(138-594) are identified at the
sides. The sample for lane 1 contained
32P-labeled DNA and unlabeled B"-(138-594). C,
gel slices containing free B" and DNA-bound B" were eluted and
processed for treatment with CNBr. The resulting
32P-labeled peptide fragments were then separated by
SDS-PAGE. The CNBr partial cleavage patterns of free and cross-linked
[32P]B"-(138-594) are shown in lanes 2 and 3. 32P-Labeled peptide
fragments are identified at the right of the panel by the
position of the cleaved methionine residue. As a control, untreated
aliquots of the free and DNA-linked [32P]B"-(138-594)
were resolved on the same SDS-PAGE and corresponded to only the free B"
(lane 1) and B"-DNA complex (data not shown),
respectively. The band marked with an asterisk (the upper
band of the doublet in lane 2) was not
consistently observed and has not been mapped. The phosphoimage density
profiles of lanes 2 and 3 are shown in
Fig. 6B.
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Figs. 6 and
7 summarize the results of this method of
mapping B" cross-links to the SNR6 gene at bp 42,
39/38, 33, 23/22, and 13/12 by partial CNBr and NTCB
(cysteine-specific) cleavage, respectively. Panel
A of Fig. 6 shows that, when B"-(138-594) is cross-linked
to the SNR6 42 probe, products of CNBr cleavage C-terminal
of Met-222 are shifted to lower mobility, implying that the B"-DNA link
is located between amino acids 223 and 276. Panel
B displays the profile of the experiment with probe
39/38 shown in Fig. 5. The Met-298 peak was consistently somewhat
reduced relative to the Met-276 peak (in seven out of seven
experiments), suggesting that a subsidiary cross-linking site of B" is
located between amino acids 277 and 297. Panel A
of Fig. 7 confirms that the bulk of the cross-linking to the 39/38
probe occurs C-terminal to Cys-280.
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Fig. 6.
Partial CNBr cleavage of
[32P]B"-(138-594) cross-linked to SNR6 photoprobes maps segments of the protein lying upstream and
downstream of the TATA box. SNR6 photoprobes 42
(A), 39/38 (B), 33 (C),
23/22 (D), or 13/12 (E) bound to TFIIIB
complexes containing [32P]B"-(138-594) were
UV-irradiated and processed as described in Fig. 5A and
under "Materials and Methods." Each panel shows the phosphoimage
density profile of the CNBr-cleavage products of the free and
DNA-linked [32P]B"-(138-594) (thin and
thick lines, respectively), normalized at the
smallest 32P-labeled polypeptide. Each peak is marked to
correspond to the methionine cleavage that generated it.
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Fig. 7.
Partial NTCB cleavage of
[32P]B"-(138-594) cross-linked to SNR6 supports the CNBr peptide mapping of B"-(138-594).
SNR6 photoprobes 39/38 (A), 33
(B), or 13/12 (C) bound to TFIIIB containing
[32P]B"-(138-594) were processed as described in Fig.
5A; gel-isolated free and DNA-linked
[32P]B"-(138-594) were reacted with NTCB and cleaved as
described under "Materials and Methods." The
32P-labeled peptide fragments of the free and cross-linked
[32P]B"-(138-594) were then resolved by SDS-PAGE and
analyzed as described for Fig. 6. Phosphoimage density profiles of the
NTCB cleavage products of free and DNA-linked
[32P]B"-(138-594) (thin and thick lines, respectively), are normalized at the smallest
32P-labeled polypeptide.
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Panel C of Fig. 6 shows that shifting the site of
DNA cross-linking downstream by only 5 bp, to bp 33, shifts the
protein side of the cross-link far toward the C-end of B"; only CNBr
cleavage products to the carboxyl side of Met-435 are shifted to lower electrophoretic mobility (the products of cleavage at Met-425 and
Met-434 fail to separate on these gels). The corresponding NTCB
cleavage pattern (Fig. 7, panel B) shows that the
point of DNA attachment is to the amino side of Cys-485. Thus, the
cross-link to bp 33 is located between amino acids 425 and 485 of B".
The result supports the evidence presented in Fig. 3B that
the amino acid 371-594 segment of B" cross-links to bp 33 of the
SNR6 gene.
Cross-linking to the SNR6 gene at bp 23/22 and 13/12
has also been examined in this manner. In these cases, the level of
cross-linking was low enough that the background of free B" (see Fig.
5B, lane 2) makes the outcome of the
analysis more tentative. Panels D and
E of Fig. 6 display the CNBr cleavage profiles of probes
23/22 and 13/12, respectively. Both profiles imply that the
cross-linking occurs at the C-terminal side of Met-425 and/or Met-434.
Partial cleavage at cysteine indicates that at least part of the
cross-linking to bp 13/12 occurs within the amino acid 426-485
segment (Fig. 7, panel C).
The same method was used to map segments of B" that are located in
vicinity to bp 38/37, 33/32, 30, and 26 of the
SUP4 gene (in TFIIIB-DNA complexes assembled with TFIIIC and
subsequently stripped of the latter with heparin). The three
upstream-lying sites of cross-linking generated nearly identical
partial CNBr digestion profiles (Fig. 8,
panels A-C) indicating that the segment of B"
that cross-links to these three sites lies between amino acids 299 and
314. (There is an indication, at or near the limits of reliability,
that the amino acid 277-297 segment makes a small contribution to DNA
cross-linking at bp 38/37, but not at bp 30.) It is feasible for
the amino acid 299-314 segment to span 8 base pairs. However, as
proposed above, a more likely explanation is that TFIIIC positions
TFIIIB heterogeneously over the AT-rich upstream region of the
SUP4 gene promoter (20), and that the amino acid 299-314
segment generates the highest levels of cross-linking, wherever it is
located. Heterogeneous placement of TFIIIB by TFIIIC may contribute to
the less complete shift to high molecular size of partial CNBr products
with C termini between Met-315 and Met-425/M434 with probe 30
(panel C). Panel C
indicates that the photo-adduct at bp 30 primarily links amino acids
299 to 314 and, with much lower efficiency, amino acids 435 to 512;
however, the background of free B" was not insignificant with this
probe. Shifting the DNA end of the cross-link another 4 bp downstream,
to 26, changed the CNBr cleavage pattern, with the B" cross-link now
completely partitioned to the C-terminal side of Met-434.
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Fig. 8.
Partial CNBr cleavage of
[32P]B"-(138-594) cross-linked to the SUP4 gene confirms that amino acids 299 to 314 lie at the upstream end
of its DNA site(s), and maps the amino acid 425/434 to 543 segment to
the vicinity of the putative TBP site. SUP4 photoprobes
38/37 (A), 33/32 (B), 30
(C), or 26 (D) bound to TFIIIC and TFIIIB
containing [32P]B"-(138-594) were stripped with heparin
to remove TFIIIC, then UV-cross-linked and processed as described in
Fig. 5A. Phosphoimage density profiles of the CNBr-cleavage
profiles of free (thin line) and DNA-linked
(thick line) [32P]B", normalized at
the two smallest peptides, are shown.
|
|
| |
DISCUSSION |
The Alignment of B" in the TFIIIB-DNA Complex--
B" is, to
varying degree, accessible to cross-linking by ABdUMP placed
site-specifically along the length of the TFIIIB-DNA complex (16).
However, it is most efficiently cross-linked to DNA upstream of the
TATA box (7). B" binding to the TBP-Brf-DNA complex extends the DNA
footprint ~10 bp upstream of the TATA box, and is stabilized by an
additional 15-20 bp DNA stretch upstream of the TATA box (see Ref. 22
and Footnote 2). Thus, the DNA segment
that is additionally covered when B" adds to the TBP-Brf-DNA complex
harbors a site of efficient B" cross-linking and contributes to the
stability of the TFIIIB-DNA complex.
A central ~225-amino acid segment of B" appears to encompass its
functional core. Two domains, one at each end of this core region
(amino acids 272-292 and 424-449), are required for
TFIIIC-dependent transcription in vitro, and one
or the other of these segments is required for TFIIIC-independent
transcription (11). An additional and partly overlapping ~65-amino
acid segment (amino acids 355-421) is required for transcription of
linear DNA (17).
The principal target for DNA cross-linking of B" lies between amino
acids 277 and 315, i.e. it encompasses the N-proximal "essential" amino acid 272-292 segment. This segment of B" faces the upstream part of the DNA site occupied by TFIIIB. Thus, the DNase I
footprint extension that is generated when B" enters the B'-DNA complex
is at least partly due to a direct B"-DNA interaction.
The N-proximal 223 amino acids of B" are not essential for
transcription of bare DNA (11). This segment of B" diminishes DNA-protein cross-linking upstream of the TATA box at least 2-fold (Table I), as though it formed an obstruction of the corresponding DNA-interacting segments of B". Deleting B" amino acids 1-185 has the
same effect on cross-linking (Table I). A segment of B" extending
approximately from amino acid 190 to amino acid 210 becomes more
accessible to cleavage by hydroxyl radical upon entry into the
TFIIIB-DNA complex (11). This is consistent with the notion that DNA
access of B" might be blocked by its internal folding, and uncovered
upon formation of the TFIIIB-DNA complex. The 137 N-terminal amino
acids of B" are also removed in making 32P-end-labeled B".
This may inadvertently facilitate the mapping of protein segments
cross-linking to specific DNA sites upstream of the TATA box by
generating higher efficiencies of cross-linking. However, if such an
effect exists, it is quantitative rather than qualitative, since
cross-linking of 32P-labeled full-length B" to the
SNR6 39/38 probe and the SUP4 38/37 probe
likewise mapped to amino acids 299-315 (data not shown).
The disposition of B" relative to DNA has been most clearly mapped in
the SNR6 gene-TFIIIB complex, as detailed below.
1) The amino acid 223-275 segment cross-links to bp 42.
2) The bp 39/38 site principally cross-links to the amino acid
299-315 segment, but some cross-linking to amino acids 277-297 is
consistently observed (Fig. 6). Deleting amino acids 272-292 also
diminishes cross-linking of B" to the bp 39/38 site (Table I).
3) Moving just 5 (or 6) bp toward the transcriptional start, to bp
33, shifts the site of the B"-DNA cross-link to the amino acid
435-485 segment. Thus, the intervening amino acids 316-434 must be
positioned away from DNA. This part of B" includes the amino acid
355-421 segment, which is required for transcription of linear DNA,
and the amino acid 424-449 segment, which is required for
TFIIIC-dependent transcription; it extends into the amino acid 390-470 "region I" segment, which is protected from hydroxyl radical cleavage upon forming the TFIIIB-DNA complex (11). Since the
amino acid 316-434 segment appears not to lie close to DNA, protection
of region I from hydroxyl radical cleavage must be due to
protein-protein interaction. A recently sequenced S. pombe open reading frame (NCBI accession CAA22645) considered to be a
candidate homologue of S. cerevisiae B" has 31% identical
(65% homologous) sequence in the region I segment.
4) Cross-linking to B" downstream of the SNR6 TATA box
principally involves the C-proximal amino acid 371-594 segment (Fig. 3B); the cross-link appears to be located C-terminal to
amino acid 425 (Fig. 6) and N-terminal to amino acid 487 (Table I), but
more precise mapping has proved inconclusive (Fig. 7). This part of B"
may not be uniquely positioned relative to DNA.
5) When B" enters into the TFIIIC-B'-DNA complex, it enhances DNase I
cleavage around the start site of transcription and increases DNase I
protection between bp 14 and 30 attributable to the B' complex
(11). Upon addition to a TFIIIC-independent B'-DNA complex, B"
generates markedly increased protection between bp 21 and 12 from
MPE-Fe(II) cleavage (23) and de novo protection between bp
22 and-18 from hydroxyl radical cleavage (22). The weak cross-linking
we have observed between B" and sites downstream of the TATA box
appears to map to the C-terminal amino acid 426-594 segment of B".
However, removal of most of this segment (amino acids 465-594) has no
effect on the TFIIIB footprint (11). This could imply that, if B"
directly generates the B"-dependent protection downstream
of the TATA box, this interaction involves the amino acid 426-464
segment, presumably through the minor groove, since cross-linking to
the major groove-protruding photoreactive side chain of ABdUMP is weak
(2). The decrease in cross-linking of B"424-438 with
SNR6 probe 13/12 might, accordingly, reflect the
disposition of this segment of B" 10 bp downstream of the TATA box.
Alternatively, and more probably, the downstream protection generated
by B" entry results from a change in the path of DNA that brings Brf
into closer proximity with downstream DNA sequence. This may
predominantly be the result of B"-Brf interactions that constrain the
path of DNA within the TFIIIB-DNA complex.
Alternative Placements on the SUP4 Gene--
The fact that
TFIIIC-directed transcription of the SUP4 gene generates
secondary start sites at +4 and +8 suggests that TFIIIB may not be
placed at a unique location on this promoter by TFIIIC (20). A
comparison of the B" mapping experiments in Figs. 6 and 8 is consistent
with this interpretation. The amino acid 299-314 segment principally
cross-links to bp 38 and-37 of the SUP4 gene with a small
contribution to cross-linking from amino acids 277-297. This is what
is also seen at bp 39 and 38 of the SNR6 gene. However,
the amino acid 299-314 segment also cross-links to the SUP4
promoter at bp 33 and 32, and makes the major contribution to
cross-linking at bp 30. The amino acid 435-512 segment makes no
significant contribution to cross-linking at bp 33/32, may make a
minor contribution to cross-linking at bp 30, and only makes the
major contribution further downstream at bp 26.
The B"-Brf Interface--
The ability of DNA with more than one
photoactive nucleotide to form multiple cross-links has been exploited
in mapping the interface of B" and Brf. Detailed analysis (Fig. 4)
shows that a high molecular weight DNA-protein adduct formed with the
SUP4 38/37 probe contains both B" and Brf. High
molecular weight bands were also generated at other sites by DNA probes
with two neighboring ABdUMP residues. We conclude that B" and Brf share an extensive interface along the length of the TFIIIB-DNA complex. This
is consistent with evidence that each half of Brf is able to recruit B"
to a TFIIIB-DNA complex (7). The N-terminal half of Brf brings the
amino acid 291-310 segment of B" into close proximity to DNA upstream
of the TATA box, and the C-terminal half of Brf brings B" (presumably
the amino acid 426-487 segment) into less close DNA proximity
downstream of the TATA box. It is the N-terminal half of Brf that
dominates Brf cross-linking downstream of the TATA box in the
TFIIIB-DNA complex, but this cross-linking requires both B" and the
C-terminal half of Brf (7). This suggests that DNA protection (from
DNase I) downstream of the TATA box is directly provided by the
N-terminal half of Brf, held in place by B" and the C-terminal half of Brf.
This inquiry was partly motivated by the observation that
B"-(263-594), B"272-292, and B"409-421 generates start
site-proximal aberrations in the TFIIIB-TFIIIC-DNA complex footprint,
with B"272-292 and B"409-421 generating identical effects (11).
One interpretation of this observation is that regions I and II of B"
are situated in close proximity to each other (11). The cross-linking
of these two regions of B" to DNA sites that are separated by only 5 or
6 bp (Fig. 6) supports this contention. Additional cross-linking of the
amino acid 426-487 segment of B" to sites downstream of the TATA box
suggests that TFIIIB bends DNA (23) so as to bring the two flanks of
the TATA box closer together. Somewhat depressed cross-linking
downstream of the TATA box is seen for B"263-594, B"424-438, and
B"409-421 (Table I). This may reflect the proximity of these two
parts of B" within the TFIIIB-DNA complex and subtle alterations in the
path of DNA when these regions of B" are deleted.
| |
ACKNOWLEDGEMENTS |
We are grateful to A. Grove, S. Kolesky, M. Ouhammouch, C. Brent, and G. A. Letts for advice and technical
help, and A. Grove for helpful comments on the manuscript.
| |
FOOTNOTES |
*
This work was supported in part by a grant from NIGMS,
National Institutes of Health.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.
Trainee of an National Institutes of Health Training Grant in Cell
Biology, Molecular Biology and Genetics. To whom correspondence may be
addressed: Dept. of Biology and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA
92093-0634. E-mail: alojipan@biomail.ucsd.edu.
§
To whom correspondence may be addressed: Dept. of Biology and
Center for Molecular Genetics, University of California, San Diego,
9500 Gilman Dr., La Jolla, CA 92093-0634. E-mail: gak@ucsd.edu.
2
A. Kumar and G. A. Kassavetis, unpublished observations.
| |
ABBREVIATIONS |
The abbreviations used are:
pol, polymerase;
TF, transcription factor;
TBP, TATA-binding protein;
bp, base pair(s);
PAGE, polyacrylamide gel electrophoresis;
NTCB, 2-nitro-5-thiocyanobenzoic acid;
ABdUMP, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-deoxyuridine
monophosphate.
| |
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ABSTRACT
INTRODUCTION
