 |
INTRODUCTION |
Bacterial RNA polymerase holoenzymes locate their cognate
promoters through
subunits that recognize specific DNA motifs located upstream of transcriptional start sites.
subunits also participate in subsequent steps of the reaction sequence that generates
an RNA chain-elongating transcription complex. For example: 1)
54 is directly involved in initiating promoter opening
next to its
12 DNA-binding site (1); 2)
70
participates directly in initiating promoter opening and binds sequence-specifically to the nontranscribed strand of the upstream segment of the transcription bubble in the open promoter complex (2-5); and 3)
70 also participates in promoter-proximal
pausing events that are required for assembly of promoter-specific
termination-resistant elongation complexes (6-8). In addition,
70 and
54 also mediate effects of
proteins that activate transcription by allowing or accelerating
promoter opening (9, 10).
Eukaryotic nuclear RNA polymerases locate their promoters through
DNA-bound complexes of core transcription initiation factors. Yeast RNA
polymerase (pol)1 III is
brought to its promoters primarily by interacting with its
transcription factor TFIIIB, which is composed of subunits TBP, Brf1,
and Bdp1 (previously called B" or TFIIIB90). A homologous assembly
operates at most human pol III promoters, whereas a paralogous assembly
containing Brf2 (previously called BRFU or TFIIIB50) in place of
Brf1 functions at a specialized subgroup of promoters that require
participation of the SNAPC DNA-binding complex and its
activators (reviewed in Refs. 11-13; see Ref. 14 for a new TFIIIB
subunit nomenclature).
TFIIIB also participates in two post-recruitment steps of
transcriptional initiation. The first evidence for this
protein-like role of TFIIIB came from analysis of the properties of
certain Brf1 and Bdp1 mutants that are inactive in transcription of
linear DNA, although they recruit pol III to the promoter (15, 16): 1)
A cluster of Bdp1 internal deletions interferes with initiation of DNA
strand separation of the upstream end of the transcription bubble; this
defect can be repaired by unpairing a short (3 or 5 bp) DNA segment at
the corresponding location, 5-9 bp upstream of the transcriptional
start site. 2) An N-terminal Brf1 deletion that removes its
zinc-binding region interferes with propagation of DNA strand
separation past the transcriptional start site; this defect is repaired
by unpairing short DNA segments of the downstream end of the
transcription bubble (17). Breaks in either DNA strand in the vicinity
of the transcriptional start also restore some transcriptional activity
to these defective TFIIIB assemblies in a strand break
location-specific and mutation-specific pattern. Interpretation of
these effects of DNA breaks leads to an explanation of how Brf1 and
Bdp1 help to enforce the upstream
downstream polarity of promoter
opening (18).
Although the demonstration of a role for TFIIIB in promoter opening
exposes a fundamental parallel between the action of initiation factors
in bacterial and eukaryotic transcription, the distinctive architecture
of bacterial and eukaryotic promoters may dictate corresponding
differences of initiation mechanism. In particular, DNA strand
separation at bacterial promoters initiates within or immediately
adjacent to the transcriptional start-proximal DNA-binding sites of
their
subunits. On the other hand, the DNA-anchoring site of TFIIIB
is centered ~17 bp upstream of the upstream edge of the transcription
bubble, and pol II as well as pol I promoter complexes are also
DNA-anchored at some distance from (and primarily upstream of) the
transcription bubble. Thus, it is not clear whether the TFIIIB subunits
should be expected to interact directly with the pol III transcription
bubble, as
70 does, or whether they might act
allosterically, perhaps through pol III subunits C82, C34, or C31,
which have a clear role in open complex formation and in initiation of
transcription (19-24). We report the outcome of experiments that
approach this question by probing DNA proximity of TFIIIB in the DNA
segment of the transcription bubble by site-specific photochemical
cross-linking.
 |
EXPERIMENTAL PROCEDURES |
DNA Templates and Probes--
The 198-bp (
60 to +138)
pU6RboxB-derived transcription template has been described
(25). Fully duplex and heteroduplex bubble-containing 86-bp (
56 to
+30) photochemical cross-linking probes all differ from
pU6RboxB at three positions: an upstream (
29) A
G
substitution to generate a TGTA mutant TATA box for TFIIIB binding in a
single orientation with the TBP variant, TBPm3 (26) and two downstream
(+8 and +9) A
T substitutions (Fig. 1A). These substitutions were
generated by PCR with mutagenic primers using the above-specified
198-bp transcription template (25) and variants of this template that
were previously used for making transcription templates with short
heteroduplex bubbles (27). Bottom (transcribed) strands that served as
templates for primer-directed incorporation of the photoreactive
deoxyribonucleotide 5-(N'-[p-azidobenzoyl]-3-aminoallyl)-dUTP
(28) with adjacent 32P-labeled deoxyribonucleotides (essentially as described in Ref. 29) and heteroduplex
formation were generated by PCR in which the primer for the bottom
strand contained a ribonucleotide at its 3' end for subsequent alkaline
hydrolysis and denaturing gel isolation (25). One half of each duplex
photoreactive probe was denatured at 94 °C in the presence of a
3-fold molar excess of bottom strands altered in sequence to form
either a
9/
5 or a +2/+6 heteroduplex bubble (Fig. 1A),
followed by annealing for 60 min at 63 °C in 30 mM
Tris-Cl, pH 8.0, 7 mM MgCl2, 50 mM
KCl, and 2 mM
-mercaptoethanol. Both duplex and
heteroduplex probes were purified on MDE-Hydrolink (BioWhittaker) gels
(30).

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Fig. 1.
Wild type Bdp1 and Brf1 are located in the
vicinity of the transcription bubble in promoter complexes containing
FLAG-tagged pol III. A, constructs for analysis of
cross-linking. U designates the photoactive T analogue
ABdUMP, and lowercase letters designate
-32P-labeled nucleotides. B, cross-linking to
the nontranscribed strand at bp 8/ 7. Each set of five
lanes shows one analysis of a promoter complex assembled
with TFIIIB only (no pol III; lanes 1-5); B' (Brf1 and
Tbp1; no Bdp1) and pol III (lanes 6-10); and TFIIIB
together with pol III (lanes 11-15). L is a
duplicate of the reaction mixture applied to immobilized anti-FLAG
antibody, FT is the flow-through; W3 and
W4 are wash fractions, and E is the eluate
released with FLAG peptide. The same proportion of the total material
in each fraction has been loaded on the gel. Cross-linked proteins are
identified at the right side. The asterisks next
to lanes 6 and 7 mark unknown materials whose
mobilities do not match Bdp1 or C128. C, cross-linking to
the nontranscribed strand at bp 8/ 7 in a preformed bubble segment
covering bp 9 to 5. Fractions and cross-linked proteins are
indicated as in panel B, except that L represents
one-half of the column load. D, cross-linking to the
nontranscribed strand at bp 8/ 7 at 30 and 0 °C. TFIIIB-DNA
complexes (lanes 1 and 3) or control reactions
lacking TFIIIB (lanes 2 and 4) were formed as
described under "Experimental Procedures" and transferred to
30 °C (lanes 1 and 2) or 0 °C (lanes
3 and 4) after the addition of nonspecific competitor
(salmon sperm) DNA. Pol III was added to each reaction mixture 5 min
later, and the incubation was continued for 20 min followed by UV
irradiation at the same temperature. Each lane shows cross-linking in
the fraction eluted from immobilized anti-FLAG antibody with FLAG
peptide. E, cross-linking to the nontranscribed strand at bp
+2/+3 in intact duplex DNA and in DNA with a preformed bubble segment
covering bp +2 to +6. The fractions and cross-linked proteins are
identified as in C.
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Proteins--
The purification and quantification of proteins
(with the exception of FLAG epitope-tagged pol III) has been described
(17). Quantities of pol III are specified as fmol of enzyme active for specific transcription (31); quantities of other proteins are specified
as fmol of protein. Wild type TBP and full-length Bdp1 were estimated
to be nearly 100% active, and full-length Brf1 was estimated to be
20% active in the formation of heparin-resistant TFIIIB-DNA
complexes. Brf1
366-409 (rtBrf1), the parental reference type for
Brf1N
68, is 70% more active than Brf1 in specific transcription.
Saccharomyces cerevisiae strain NZ16 (32) (originally
constructed by Nick Zecherle and Benjamin Hall, University of
Washington) was generously provided by Blaine Bartholomew (Southern
Illinois University at Carbondale). The strain contains a hexahistidine tag followed by four tandem FLAG epitope tags at the N terminus of the
RET1 gene (encoding the C128 subunit of pol III). A rapid purification scheme involving tandem chromatography on BioRex 70, nickel-nitrilotriacetic acid-agarose, and heparin-Sepharose, using an
automated fast protein liquid chromatography system, yielded apparently
homogeneous pol III (as judged by silver staining) with 40% of
molecules active for specifically initiating transcription. The details
of the procedure and chromatography program are available upon request.
Photochemical Cross-linking--
Preinitiation complexes were
prepared in two closely related ways. In procedure I (Figs. 1,
B, C, and E, and
2) TFIIIB-DNA complexes were formed
during a 60-min incubation at 20 °C in 96 µl of binding buffer (40 mM Tris-Cl, pH 8.0, 7 mM MgCl2, 60 mM NaCl, 3 mM
-mercaptoethanol, 5 µg/ml
poly(dG-dC):poly(dG-dC), 25 µg/ml bovine serum albumin, 5% (v/v)
glycerol) containing 1.6 pmol of TBPm3, 1.4 pmol of Brf1, or 1 pmol of
Brf1N
68, 0.9 pmol of Bdp1, or Bdp1
355-372, and 24 fmol of
photoreactive DNA probe. This was followed by the addition of 600 ng of
sheared salmon sperm DNA in 4 µl of diluent buffer (10 mM
Tris-Cl, pH 8, 0.1 mM Na3EDTA) and 9.6 fmol of
FLAG-tagged pol III in 24 µl of buffer H (20 mM NaHEPES,
pH 7.8, 20% (v/v) glycerol, 7 mM MgCl2, 0.1 mM Na3EDTA, 10 mM
-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride,
1 µg/ml leupeptin and pepstatin) also containing 65 mM
(NH4)2SO4, 100 mM NaCl,
and 100 µg/ml bovine serum albumin for 20 min. Complexes were
UV-irradiated as described (33), loaded onto 20-µl anti-FLAG M2
agarose columns equilibrated in buffer A (20 mM Tris-Cl, pH
8.0, 7 mM MgCl2, 100 mM NaCl, 10 mM
-mercaptoethanol, 20 µg/ml bovine serum albumin,
5% (v/v) glycerol, 1 mM Na3EDTA, 0.1% Tween
20, and 0.5 mM phenylmethylsulfonyl fluoride), washed four
times with 40-µl aliquots of buffer A and eluted with 40 µl of
buffer A containing 100 µg/ml 3× FLAG peptide. Nuclease treatment
followed (33). In procedure II (Fig. 1D) TFIIIB-DNA complexes were formed for 40 min in 40 µl of the above binding buffer
containing 90 mM instead of 60 mM NaCl, 1.8 pmol of TBPm3, 200 fmol of Brf1, 300 fmol of Bdp1, and 12 fmol of
photoreactive DNA probe. This was followed by the addition of 450 ng of sheared salmon sperm DNA in 3 µl of diluent buffer and 28 fmol
of FLAG-tagged pol III in 8 µl of buffer H also containing 50 mM (NH4)2SO4 and 100 µg/ml bovine serum albumin for 20 more min prior to UV irradiation. Chromatography on anti-FLAG agarose proceeded as above. All of the
experiments shown were replicated two or three times with essentially
identical results.

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Fig. 2.
Effects of Brf1 and Bdp1 deletions on
cross-linking to DNA upstream and downstream of the transcriptional
start site. A and B present cross-linking to
the nontranscribed strand at bp 8/ 7 and +2/+3, respectively. Each
lane shows cross-linking in the fraction eluted from
immobilized anti-FLAG antibody with FLAG peptide (corresponding to
fraction E, Fig. 1B, lane 15) for samples
assembled and analyzed as shown in detail in Fig. 1. DNA templates and
components of TFIIIB are indicated above each
lane. Cross-linked proteins are identified at the sides. The
amino acid 1-68 deletion in Brf1 allows a clear separation from C82,
whereas the amino acid 355-372 deletion in Bdp1 does not change
electrophoretic mobility.
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 |
RESULTS |
Access of Brf1 and Bdp1 to the Opening DNA Bubble of the
Transcription Initiation Complex--
If Bdp1 and Brf1 play a direct
role in DNA melting at the transcriptional start, they should contact
this DNA segment. Both TFIIIB subunits can be cross-linked to both DNA
strands near bp +1 in TFIIIB-DNA complexes (15, 32, 34-36), but it
remained to be determined whether the proximity of Bdp1 and Brf1 to the start site of transcription is maintained upon entry of pol III into
the initiation complex or whether these subunits of TFIIIB are
displaced. The following approach was used to examine the cross-linking
of TFIIIB in the presence of DNA-bound pol III. Initiation complexes
were assembled with FLAG-tagged pol III on duplex DNA as well as DNA
unpaired at the upstream end (bp
9 to
5) or downstream end (bp +2
to +6) of the transcription initiation bubble. Duplex DNA and bp
9/
5 "bubble" DNA were photoactively tagged in the
nontranscribed strand at bp
8 and
7 with ABdUMP. Duplex and
bp +2/+6 bubble DNA were also photoactively tagged in the
nontranscribed strand at bp +2 and +3. Each DNA probe contained a
radioactive nucleotide next to its photoactive nucleotides (Fig. 1A). TFIIIB-pol III complexes assembled on these DNAs were
UV-irradiated, pol III-containing complexes were separated from
TFIIIB-DNA complexes on immobilized anti-FLAG antibody, and both the
antibody-retained and flow-through materials were analyzed to identify
cross-linked proteins. Control promoter complexes were made with TFIIIB
alone, with the omission of Bdp1 and with promoter opening-defective TFIIIB assembled with Bdp1
355-372 or Brf1N
68. The nearly
symmetric U6 TATA box allows some TBP binding and resultant assembly of TFIIIB-pol III complexes in the reverse orientation, which complicates structural interpretation of cross-linking. This problem was
circumvented by the use of the mutant TBPm3 in conjunction with a TGTA
box promoter (TGTAAATA) (26, 37). Truncating the upstream end of the
DNA probe at bp
56 also eliminated the possibility of pol III
binding to reverse-oriented TFIIIB-DNA complexes.
The principal result of the analysis of probes photoactively tagged at
bp
8 and
7 was that both Bdp1 and Brf1 (the latter cross-linking
more weakly) are located in the vicinity of DNA in a pol III-containing
complex (Fig. 1B, lane 15). TFIIIB complexes were
cleanly separated from TFIIIB-pol III-DNA complexes on the anti-FLAG
column (lanes 1-5 and 11-15); omission of Bdp1 prevented pol III recruitment (lanes 6-10). The C160, C128, C82, and
C34 subunits of pol III were also cross-linked to DNA at this location.
Preopening bp
9 to
5 restores the transcriptional activity of
promoter opening-defective Bdp1 internal deletion mutants. Completely
opening bp
9 to
5 by unpairing clearly correlated with greatly
increased cross-linking of the C82 pol III subunit in the presence of
full-length Bdp1 (Fig. 1C, lane 7; compare the
ratio of the C82:Brf1 signals with Fig. 1B, lane
15). Although this result appears to imply that cross-linking of
the C82 subunit at positions
8/
7 is an inherent property of open
complex formation, this may not be the case. Wild type TFIIIB-pol
III-duplex DNA complexes formed and UV-irradiated at 30 °C (open)
and 0 °C (closed) both displayed equivalent, low levels of C82
cross-linking relative to Bdp1, Brf1, and the other subunits of pol III
(Fig. 1D, compare lanes 1 and 3; only
the FLAG peptide-eluted fractions are shown). The somewhat weaker
cross-linking of all proteins in Fig. 1D (lane 3, relative
to lane 1) was not reproducibly observed and probably reflects variations in the efficiency of pol III assembly at 0 °C or
in recovery of complexes from anti-FLAG agarose, because the relative
efficiencies of all protein cross-linking (including C34; not shown) at
30 and 0 °C were indistinguishable. Cross-linking to C160 and C128
(relative to Bdp1 and Brf1) also increased when the bp
9/
5 segment
was preopened but did not change between 30 and 0 °C in complexes
formed with duplex DNA.
Cross-linking at the downstream segment of the transcription bubble was
also examined. Preopening bp +2 to +6 restores transcriptional activity
to TFIIIB assembled with the N-terminal deletion subunit Brf1N
68.
Full-length Bdp1 and Brf1 were found to cross-link to the
nontranscribed strand at bp +2/+3 in duplex DNA along with C128 (Fig.
1E, lane 10) and C34 (not shown). Cross-linking
of the largest pol III subunit, C160, at this location was very weak. Opening the bp +2/+6 bubble greatly increased the efficiency of Brf1
cross-linking and also increased Bdp1 cross-linking relative to C128
(lane 5). Pol III entry into the bp +2/+6 bubble promoter changed the relative efficiencies of Brf1 and Bdp1 cross-linking (compare lanes 2 and 5). Cross-linking of Brf1 and Bdp1 to
DNA at bp +2/+3 does not require promoter opening, because it was also
observed in FLAG antibody-purified TFIIIB-pol III promoter complexes
formed and UV-irradiated at 0 °C and because the relative cross-linking efficiencies of C160, C128, Bdp1, Brf1, and C34 at 30 and
0 °C were indistinguishable (data not shown).
The above results indicate that Bdp1 and Brf1 lie in close proximity to
the upstream and downstream borders of the prospective and actual open
complex. Because the photoreactive moiety of ABdUMP barely protrudes
beyond the edge of the major groove (28), both subunits could
potentially play a direct role in the nucleation of DNA strand opening
and downstream propagation of the bubble. Small (~15 amino acids)
deletions between amino acids 355 and 421 of Bdp1 prevent the onset of
DNA strand separation at the upstream edge of the prospective
transcription bubble. Deletion of the N-terminal 68 amino acids of Brf1
prevents the downstream propagation of the transcription bubble. The
effect of promoter opening-defective Bdp1 and Brf1 deletion mutants on
the cross-linking of TFIIIB and pol III subunits near the start site of
transcription was examined next (Fig. 2). Protein-DNA complexes
containing both pol III and TFIIIB were affinity-purified after UV
irradiation, as in the preceding experiment. Only the FLAG
peptide-eluted fractions are shown.
TFIIIB-pol III-DNA complexes assembled with Bdp1
355-372 displayed
significantly diminished cross-linking of Bdp1, Brf1, and the C160,
C128, and C82 subunits of pol III relative to the C34 subunit of pol
III to bp
8/7 in duplex DNA (Fig. 2, compare lanes 1 and
2). A similar diminution of C160 and C128 cross-linking was
previously observed with Bdp1
372-387 in a mixture of TFIIIB- and
TFIIIB-pol III-DNA complexes (15). Preopening the bp
9/
5 bubble,
which allows a fully open complex to be formed with Bdp1
355-372, partially restored C160, C128, Bdp1, and Brf1 cross-linking relative to
C34 (compare lanes 5 and 2). The effect of
deleting Bdp1 amino acids 355-372 on cross-linking at the downstream
end of the transcription bubble (probe +2/+3) was more pronounced, with
a nearly complete loss of cross-linking of Bdp1, Brf1, and the C128
subunit of pol III but not of C34 cross-linking (compare lanes
7 and 8). The loss of pol III large subunit
cross-linking to duplex DNA evidently reflects a subtle structural
change, in light of a prior methidiumpropyl EDTA-Fe(II)
footprinting analysis of the transcribed strand of TFIIIB-pol III-DNA
complexes formed with wild type Bdp1 and Bdp1
372-387 (17) showing
identical protection of duplex DNA in this region. Opening the bp +2/+6
bubble, which does not ameliorate the promoter opening defect of
Bdp1
355-372 (17), partly restored C128, Brf1, and Bdp1
cross-linking (compare lanes 11 and 8).
Deletion of the N-terminal 68 amino acids of Brf1 had nearly the
opposite effect, in that cross-linking of the C34 pol III subunit at bp
8/
7 in duplex DNA decreased significantly relative to that of Bdp1,
Brf1, C160, and C128 (Fig. 2, compare lanes 1 and
3). The most pronounced effect of the N
68Brf1 deletion on TFIIIB-pol III complexes was the loss of cross-linking of the C82
subunit of pol III (lane 3). Opening the bp
9/
5 bubble
restored cross-linking to C82 and increased cross-linking to C34
(lane 6). The pol III complex assembled on bp
9/
5 bubble
promoters with Brf1N
68 remains defective in downstream bubble
propagation and transcription (17), so restoration of C82 cross-linking to bp
8 and
7 only reflects local strand separation rather than transcriptional competence. Brf1N
68 had little effect on the pattern
of cross-linking at downstream segment of the transcription bubble
(probe +2/+3; lanes 9 and 12).
In summary, we conclude that Bdp1, Brf1, and four pol III subunits lie
close to the upstream edge of the transcription bubble both in open and
in closed promoter complexes. One signature of this segment of the open
complex transcription bubble is cross-linking of the C82 pol III
subunit, which increases when this region is preopened and dramatically
decreases in complexes formed with promoter opening-defective
Brf1N
68. Proximity of Brf1 and Bdp1 to DNA extends downstream of the
transcriptional start site, in both open and closed pol III-promoter
complexes. The characteristic of the downstream site is the loss of
cross-linking of Bdp1, Brf1, and the C128 pol III subunit in complexes
formed with Bdp1
355-372. The regions in Bdp1 and Brf1 that
cross-link to the nontranscribed strand of the transcription bubble lie
outside of the protein segments whose deletion prevents open complex
formation (that is, outside Bdp1 amino acids 355-421 and Brf1 amino
acids 1-68; data not shown).
 |
DISCUSSION |
Implications of the Complexity of Protein Cross-linking at 30 and
0 °C--
Four subunits of pol III (C160, C128, C82, and C34),
Bdp1, and Brf1 all lie close to bp
8 and
7 on the nontranscribed
strand at the upstream end of the open complex transcription bubble. At
bp +2 and +3, at the downstream end of the open complex transcription bubble, cross-linking of C160 diminished significantly, and
cross-linking of C82 was not detected. This pattern of cross-linking is
similar to what has been observed previously on the SUP4
tRNA and 5 S rRNA genes, with the exception that cross-linking to Bdp1
and Brf1 was either barely detectable or absent downstream of bp +1 in
the presence of TFIIIC but could be detected at bp +6 when TFIIIC was
removed (Ref. 34; see Ref. 32 for a comprehensive survey of protein
cross-linking between bp
17 and +17). The proximity of these pol III
and TFIIIB subunits to these sites was not strongly dependent on open
complex formation; the same subunits were cross-linked at 30 and
0 °C. The close proximity of the nontranscribed strand to multiple
protein subunits near the start site of transcription has also been
observed in human pol II initiation complexes: Rpb1, Rpb2, TFIIB, and
the RAP74 subunit of TFIIF co-cross-link at the upstream edge of the
prospective transcription bubble in both closed and open promoter
complexes; Rpb1, Rpb2, Rpb5, TFIIE-
, and the ERCC3 subunit of TFIIH
co-cross-link at the downstream edge of the transcription bubble
(38).
Two aspects of the cross-linking patterns are somewhat puzzling and
deserve comment. First, it is possible to rationalize the cross-linking
of six subunits of pol III and TFIIIB to the
8/
7 probe (or four
subunits to the +2/+3 probe) in open promoter complexes. The
nontranscribed strand of the pol III open complex is not constrained
(18) and therefore might not be tethered to any particular site.
However, the same proteins are also cross-linked to DNA at 0 °C in
the absence of DNA strand separation. Although the ABdUMP reactive side
chain has some rotational flexibility, it barely projects beyond the
DNA major groove, making it unlikely that two adjacent side chains
would access six proteins. Heterogeneity of pol III placement at the
start site of transcription by TFIIIB would account for the diversity
of subunits cross-linked at 0 °C. This possibility clearly applies
to the previously analyzed TATA-less SUP4 tRNA gene, for
which alternative placements of TFIIIB by TFIIIC lead to heterogeneity
in selection of the transcriptional start site (39, 40). However, this
does not appear to be the case for the TATA-containing SNR6
(U6) gene; initiation occurs exclusively at bp +1 on U6 DNA templates
with an intact transcribed strand (25).
We suggest instead that the polymerase-DNA association in the
TFIIIB-pol III-promoter closed complex is transient, like that of
bacterial RNA polymerase closed complexes. Transient release and
reassociation of duplex DNA with pol III would account for the
diversity of cross-linking observed at 0 °C. We note that the DNase
I and methidiumpropyl EDTA-Fe(II) footprints of gel-purified TFIIIB-pol III-DNA complexes are less complete for pol III than for
TFIIIB (Fig. 4c in Ref. 15, Fig. 7 in Ref. 27, and Fig. 8 in
Ref. 17), an observation that would also be accounted for by a loosely
held pol III-DNA interaction. Transient association of DNA may also
partly account for the cross-linking of nine proteins to ABdUMP at bp
5 on the transcribed strand in pol II preinitiation complexes (Ref.
41; although partial complex assemblies may exist in this study, three
subunits of the final member of this complex, TFIIH, cross-link to this position).
The second peculiarity concerns the absence of strong differences
between the patterns of cross-linking at 0 and 30 °C. One anticipates that the pattern of cross-linking at bp +2/+3 would change
when the promoter opens because a fully opened promoter complex pulls
an additional 4-5 bp of downstream DNA into polymerase (25), and open
complex formation should accordingly shift the location of bp +2 and +3
relative to protein. In contrast, we found no significant difference in
the relative levels of cross-linking of C160, C128, Bdp1, Brf1, and C34
at 30 and 0 °C (data not shown). However, preopening the downstream
end of the transcription bubble between bp +2 and +6 significantly
enhanced +2/+3 probe cross-linking of Brf1 relative to C128 (Fig.
1E). A prior analysis of the SUP4 tRNA and 5 S
rRNA genes suggested that cross-linking of the C82 subunit of pol III
might also depend on open complex formation (35). In that work, C82 was
cross-linked to DNA containing the photoreactive nucleotide
4-S-thymidine upstream of the start site of transcription
but not with ABdUMP at the same positions. Efficient cross-linking to
4-S-thymidine, like its RNA counterpart,
4-S-uridine, occurs at sites that become unpaired or
helically distorted (42-46). Again, no difference in the relative
amounts of C82 cross-linking at bp
8/
7 was observed between 0 and
30 °C (Fig. 1D), yet preopening the upstream end of the
transcription bubble increased the relative efficiency of C82
cross-linking ~4-fold (Fig. 1C).
The simplest interpretation of the above observations is that even at
30 °C, the TFIIIB-pol III-DNA complex is predominantly closed. If
the 4-fold difference in C82 cross-linking reflects the difference
between closed and fully open complexes, then open complex formation at
30 °C would need to be less than 20% to obscure detection of
increased C82 cross-linking. Alternatively, the changed pattern of
cross-linking that is generated by preopening different segments of the
transcription bubble may result from a fraction of these templates that
fail to enter the single-stranded binding channel of pol III. A
single-stranded bubble lying at the surface of polymerase in the closed
complex would have considerably greater reach and flexibility than
duplex DNA.
An Indirect Role for TFIIIB in Open Complex
Formation--
Deletions and point mutations in the N-terminal domain
containing the zinc ribbon motif and the adjacent sequence of both Brf1
and TFIIB display three similar properties: 1) a defect in binding
their cognate polymerases, 2) alterations in transcriptional start site
selection and 3) a defect in transcription at a step subsequent to
polymerase recruitment (16, 17, 47-52). TFIIB has also been observed
to cross-link to DNA within the transcription bubble region of the pol
II initiation complex (38). It is likely that the post-recruitment
roles of TFIIB and Brf1 are related mechanistically but not necessarily
in open complex formation.
The cross-linking experiments shown here demonstrate an intimate
proximity of Brf1 and Bdp1 to the DNA segment that becomes unwound in
the pol III open complex and also to the pol III subunits likely to be
involved in open complex formation (Fig. 1). This proximity is
consistent with a direct involvement of TFIIIB in the DNA strand
separation process during open complex formation. However, the effects
of promoter opening-defective deletion mutations in Bdp1 and Brf1 on
cross-linking do not provide additional evidence for the direct
involvement of TFIIIB. In particular, the expectation that the segments
of Brf1 and Bdp1 whose deletion prevents open complex formation would
be proximal to the prospective or actual open complex transcription
bubble is not realized (Fig. 2). Instead, the region of Bdp1 that
cross-links to the bp
8 and
7 probe has been tentatively mapped
between Met425 and Cys485, using a technique
similar to one that was described previously (Ref. 53 and data not shown).
The results of these experiments favor an indirect mode of action of
TFIIIB in open complex formation. If promoter opening-defective deletion mutations in Bdp1 or Brf1 alter or prevent DNA/protein contacts by pol III that are required for pol III-mediated DNA strand
opening, one would predict that these deletions might affect the
cross-linking of pol III subunits. This is what is observed (Fig. 2).
Promoter opening-defective Bdp1 mutations grossly alter the proximity
of pol III to DNA, reducing cross-linking of C160, C128, and C82 to bp
8 and
7 and obliterating cross-linking of all pol III subunits,
except for C34, to bp +2 and +3. Similarly, the promoter
opening-defective Brf1N
68 affects cross-linking of the C82 and C34
pol III subunits to bp
8 and
7. Thus, although the Brf1 and Bdp1
subunits of TFIIIB are located close to the transcriptional start site
in pol III-containing preinitiation complexes and can be cross-linked
from the upstream and even the downstream edge of the DNA segment that
forms the transcription bubble, the evidence of this analysis favors an
indirect, pol III-mediated mode of action of TFIIIB in promoter opening
rather than a bacterial
-like direct participation in DNA strand separation.