Department of Biology and Center for
Molecular Genetics, University of California, San Diego, La Jolla,
California 92093-0634
Received 23 April 1998/Returned for modification 26 May
1998/Accepted 3 June 1998
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INTRODUCTION |
Eukaryotic nuclear and archaeal
transcription apparatuses share an essential common feature: the RNA
polymerase (pol) is brought to the transcriptional start site by a
DNA-bound assembly of transcription initiation proteins. The
promoter-marking assembly of archaeal RNA polymerases and the core
promoter-marking assembly of eukaryotic pol II each consist of two
proteins: TATA-binding protein (TBP) and the phylogenetically related
transcription factor B (TFB) or TFIIB (for archaeal or pol II
transcription systems, respectively) (43).
The situation differs only slightly for transcription by pol III, whose
promoter-marking assembly, TFIIIB, is composed of three subunits: TBP,
Brf (named for its relatedness to TFIIB) (7, 11, 33), and B"
(18, 24). In Saccharomyces cerevisiae, the three
components of TFIIIB are held together by a more stable interaction
between Brf and TBP (which together make up the B' component of TFIIIB)
and by a weaker interaction between TBP-DNA-bound Brf and B" (17,
22); a weaker direct B"-TBP interaction has also been noted
(12, 37). Each of these TFIIIB components is required for
all pol III transcription in yeast. Homologs of Brf and B" serve as
components of the pol III system of humans (42, 44) but are
not ubiquitously required. It appears instead that specialized
alternative promoter-marking assemblies participate in different
classes of human pol III promoters (16, 34, 42, 48).
The homology relationship of Brf with TFIIB is confined to the
N-terminal half of the much larger Brf; similarities between the
C-proximal half of Brf and proteins of the pol II transcription initiation apparatus have not been discerned, at least at the level of
the primary amino acid sequence (8, 11, 44).
We and others are undertaking molecular genetic dissections of TFIIIB
(12, 21, 30, 37, 38, 40; see also references 10
and 27). The experiments presented below analyze the TFIIIB assembly properties and transcriptional capabilities of a large set of
Brf fragments. Functional Brf can be assembled from selected pairs of
these fragments, effectively splitting the protein at diverse sites and
generating internal deletions without the constraint of joining
together domains or regions of Brf that might normally be well
separated in space. The interactions of the individual fragments of
split Brf with TBP and with serial locations along the DNA-binding site
of TFIIIB have also been mapped. The results of these analyses suffice
for the specification of a global map of protein and DNA interactions
in the TFIIIB-promoter complex.
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MATERIALS AND METHODS |
DNA.
Plasmids pU6LboxB and pTZ1 have been
described elsewhere (26, 47). The 75-bp TA-30-B6 DNA probe
for electrophoretic mobility shift assay (EMSA) was generated by
annealing the two synthetic oligonucleotides shown in Fig.
1a, one of which was 5' end labeled with
T4 polynucleotide kinase and [
-32P]ATP. The 240-bp
TA-30 variant SUP4 tRNA gene probe for EMSA and
methidiumpropyl-EDTA (MPE)-Fe(II) footprinting was generated by PCR as
described previously (19, 30). The 59-bp photochemical cross-linking probes were generated essentially as described elsewhere (21) with the primers shown in Fig. 4a.
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FIG. 1.
Effects of N- and C-terminal truncations in Brf on the
TFIIIC-independent assembly of B' (Brf plus TBP)-DNA and TFIIIB-DNA
complexes. (a) The 75-bp TA-30-B6 probe for EMSA for TFIIIC-independent
assembly of TBP, Brf, and B". (b) EMSA for TFIIIB-DNA complex
formation. Ten femtomoles of the TA-30-B6 probe was incubated with 10 fmol of TBP, 50 fmol of full-length Brf or the indicated truncated Brf,
and B" (1× = 50 fmol), as indicated above each lane. Reaction
conditions were otherwise as specified in Materials and Methods, and
electrophoresis was done in Mg2+-containing native gels.
Control reactions (not shown) demonstrated that Brf-DNA complexes did
not form in the absence of TBP. TBP-DNA, B'-DNA, and TFIIIB-DNA
complexes are identified at the right.
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Proteins.
TFIIIC, pol III, recombinant TBP, recombinant
TBPm3, Brf(317-596) (the Brf fragment spanning amino acids
317 to 596), recombinant wild-type B" (native purification), and
recombinant B"(137-594) were purified and assayed as described
elsewhere (21, 25, 30, 47). Full-length Brf containing
hexahistidine tags at both N and C termini was generated and purified
as follows. The expression vector pET21b was modified by inserting
TATG(CAC)6CCCATG between the NdeI and
BamHI sites (top strand shown; the bottom strand provided
the appropriate overhangs), generating pET21b-H6-Nco with
an NcoI site now overlapping the BamHI site. The
C-terminally His6-tagged Brf gene contained on an
NcoI/BlpI fragment from pSH360 (11)
was then ligated into the equivalent sites of pET21b-H6-Nco and subsequently transformed into Escherichia coli
BL21(DE3)pLysS. Induced cells (33 g) were lysed under native
conditions as described for B"(138-594) (30). The lysate was
sedimented at 22,000 × g for 1 h, the pellet was
resuspended and solubilized in buffer A (50 mM Tris-Cl [pH 8.0],
0.1% [wt/vol] Tween 20, 7 mM 
-mercaptoethanol, 10% [vol/vol]
glycerol, 6 M guanidine HCl, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg each of leupeptin and pepstatin per ml) and then sedimented at
22,000 × g for 30 min to remove insoluble material, and the supernatant fluid was applied to a 16-ml Ni-nitrilotriacetic acid (NTA) agarose column. The column was developed sequentially with
buffers containing 100 mM
NaxHxPO4, 7 M deionized urea, -mercaptoethanol, and protease inhibitors as in
buffer A (32 ml of each) at pHs 6.7, 6.3, 5.9, 5.7, 5.5, 5.1, and 4.7, respectively. The major contaminants [Brf(165-596) and other singly
hexahistidine-tagged truncated forms of Brf] eluted at pH 5.9 and 5.7. Doubly hexahistidine-tagged Brf, which eluted at pH 5.5 and 5.1, was
adjusted to pH 7.8 with Tris base and then dialyzed for 6 to 12 h
sequentially against buffer D500 (30) with 10 µM
ZnSO4 also containing 3, 1.5, 0.75, and 0 M urea.
N- and C-terminal deletions of Brf were generated by PCR amplification
from pSH360 with a 1:1 mixture of Pfu and Taq DNA
polymerases and oligonucleotides (sequences available upon request)
that introduced NcoI sites at the N termini and
XhoI sites at the C termini for insertion into
pET21b-H6-Nco. N-terminal amino acid sequences of
full-length Brf (doubly hexahistidine tagged), Brf(1-n), and Brf(164-n) were MHHHHHHP; Brf(284-n) and
Brf(435-n) were preceded by MHHHHHHPM, and
Brf(320-n) was preceded by MHHHHHHPME. Brf C termini at
amino acids 165, 282, 320, 438, 545, and 596 were followed by an UAA
termination codon; full-length Brf had six histidine residues added at
its C terminus. The resulting plasmids were introduced into E. coli BL21(DE3) containing the argU expression plasmid
pUBS520 (32, 39), generously provided by I. Willis (Albert
Einstein College of Medicine) and R. Mattes (Max-Planck-Institut für Züchtungsforschung, Cologne, Germany). Brf fragments
for most EMSA and transcription experiments were purified from lysates of induced 50-ml cultures on Ni-NTA agarose under native conditions, as
described for B"(137-596) (30). Three Brf fragments were also purified under denaturing conditions from cells lysed by resuspension in buffer A (specified above) also containing 7 M guanidine-HCl, removal of insoluble debris, and Ni-NTA agarose chromatography, as described for doubly His-tagged Brf except that the
pH 5.9 and 5.7 elution steps were omitted. The fraction eluting at pH
5.5 was neutralized and chromatographed on Superose 12 in buffer D500
containing 7 M urea, and Brf was renatured by stepwise dialysis out of
urea, as described above for doubly His-tagged Brf. Renatured
Brf(1-282) and Brf(284-596) were used for photochemical cross-linking
analysis and renatured Brf(1-282), Brf(284-596), and Brf(1-438) were
used for some transcription assays.
Quantities of TFIIIC are specified as femtomoles of DNA-binding
activity, and quantities of pol III are specified as femtomoles of
enzyme active for specific transcription (30). B",
B"(137-594), TBP, TBPm3, and Brf proteins are specified as
femtomoles of protein (relative to a standard curve for Coomassie
blue-stained bovine serum albumin [BSA] on sodium dodecyl sulfate
[SDS]-polyacrylamide gels or determined by the predicted extinction
coefficient of the protein [for B" and TBP]). B", B"(137-594), and
TBP were estimated to be nearly 100% active, and full-length Brf was
estimated to be 20% active, in the formation of heparin-resistant
TFIIIB-DNA complexes (25).
Assays.
Protein-DNA complexes for EMSA, transcription, and
photochemical cross-linking were formed in 16- to 20-µl volumes
containing 40 mM Tris-Cl (pH 8.0), 7 mM MgCl2, 3 mM
dithiothreitol (or 3 mM 2-mercaptoethanol for photochemical
cross-linking), 4 to 6% (vol/vol) glycerol, 100 µg of BSA per ml (60 µg/ml for photochemical cross-linking), and 50 to 70 mM NaCl for
TFIIIC-independent, or 70 to 90 mM NaCl for TFIIIC-dependent, DNA
complex formation and transcription. For EMSA with the TA-30-B6 probe
(10 to 20 fmol), reaction mixtures were incubated for 60 min at 20°C
and contained 100 ng of poly(dG-dC) · poly(dG-dC), 10 to 100 fmol of TBP, 50 to 100 fmol of Brf or Brf fragment, and 50 to 300 fmol
of B", when present (ranges in amounts referring to titrations). For EMSA with the SUP4 TA-30 probe (9 fmol), reaction mixtures
were incubated for 40 min at 20°C and contained the quantities of
TFIIIC, TBP, Brf or Brf fragment, and B" noted in the appropriate
figure legend.
Protein-DNA complexes were analyzed on 4% polyacrylamide gels
containing 20 mM Tris-Cl (pH 8.0), 2 mM EDTA, and 4% (vol/vol) glycerol or containing 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 2.5 mM
MgCl2, 0.5 mM dithiothreitol, and 4% (vol/vol) glycerol.
The electrode buffers lacked glycerol and dithiothreitol and were recirculated during electrophoresis.
Reaction mixtures for transcription contained 200 ng of
pU6LboxB or pTZ1 supercoiled plasmid DNA as the template, 8 to 16 fmol of pol III, 40 to 80 fmol of TFIIIC (with pTZ1 only), 50 to
200 fmol of TBP, 150 to 300 fmol of B", and 50 to 400 fmol of Brf or
its truncated variants (the ranges for TBP and Brf refer to
titrations). Following complex formation for 40 or 60 min at 20°C, 5 µl of a nucleotide mixture providing 200 µM ATP, 100 µM CTP, 100 µM GTP, and 25 µM [
-32P]UTP (10,000 cpm/pmol) was
added for 30 min of transcription. Samples were processed for
electrophoresis on 10% polyacrylamide gels containing 8 M urea as
described previously (30). The quantified transcription
reaction mixtures represented in Tables 1 and 2 and Fig. 2 contained 50 fmol of pU6LboxB, 100 fmol of TBP, 150 fmol of B", 10 fmol
of pol III, 300 to 350 fmol of Brf (or its truncated variants), and 50 mM NaCl (120 mM NaCl with full-length Brf). Transcription was
quantified, relative to that in control reactions with intact Brf
included in each experiment, by phosphorimaging.
Reaction mixtures for photochemical cross-linking contained 10 fmol of
photoreactive DNA probe, 100 ng of poly(dG-dC) · poly(dG-dC), 400 fmol of TBPm3, 100 fmol of B"(137-594) when present,
and 150 to 300 fmol of the specified Brf fragments or 350 to 700 fmol of full-length Brf. UV irradiation and nuclease treatment prior to
electrophoresis on SDS-10 or 15% polyacrylamide gels was carried out
as described elsewhere (3). Cross-linking was quantified by
phosphorimage analysis and normalized to cross-linking of intact Brf
and B"(138-594) within the TFIIIB-DNA complex that served as the
control for each experiment.
Reaction mixtures for footprinting with MPE-Fe(II) contained, in 15 µl of buffer (20 mM Tris-Cl [pH 8], 95 mM NaCl, 3.5 mM MgCl2, 2.6 mM dithiothreitol, 100 µg of BSA per ml),
55 fmol of TA-30 variant SUP4 gene probe, 100 ng of pGEM
(carrier) DNA, 250 fmol of TBP, 500 fmol of Brf or 500 fmol each of
Brf(1-282) and Brf(284-596), as specified, 500 fmol of B", as
specified, and 62 fmol of TFIIIC. DNA cutting at 21°C was started by
adding MPE-Fe(II) to 2 µM and sodium ascorbate to 1 mM and stopped by
adding 2 µl of 50 µM bathophenanthroline and 2 µl of 40%
(vol/vol) glycerol. Reaction mixtures were resolved by electrophoresis
on nondenaturing 4% acrylamide gels as specified above and exposed
without drying to an image plate to locate the desired protein-DNA
complex. The latter was cut out of the gel and eluted into 20 mM
Tris-Cl (pH 8)-0.2% (wt/vol) SDS-0.5 mM EDTA-1 M LiCl. DNA was
extracted from the eluate with phenol-chloroform, precipitated with
ethanol, redissolved, denatured, and resolved by electrophoresis on a
DNA-sequencing (10% acrylamide) gel. Dried gels were analyzed on a
phosphorimager as described previously (30).
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RESULTS |
A Brf sequence that is required for stable interaction with the
other components of the TFIIIB-DNA complex.
In preceding work
(21), we analyzed three N-terminal deletion variants of Brf
and showed that removing 164 N-proximal TFIIB-homologous amino acids
does not abolish the ability to direct TFIIIC-independent TFIIIB-DNA
complex assembly or transcription. In fact, the C-terminal half of Brf
retains some ability to interact with TBP and with the B" component of
TFIIIB. We have now combined nested series of C-terminal and N-terminal
deletions in order to define the role of individual parts of Brf in
assembling TFIIIB-DNA complexes.
The first series of experiments examines the ability of truncated Brf
to interact with TBP and B" at a TATA box. The DNA that was used for
most of the analysis has a 6-bp TATA box, TATAAA, with
4-nucleotide loops at sites at which bound TBP kinks DNA (14, 15,
20, 28, 29). Because it forms a more stable complex with
wild-type TBP than does the corresponding perfect duplex DNA, this
loop-containing DNA (designated TA-30-B6 [Fig. 1a]) facilitates
analysis of B' (i.e., TBP plus Brf)-DNA complexes by gel
electrophoresis. At the same time, the loop-containing TA-30-B6 DNA
probe retains specificity of interactions, in the sense that DNA
binding of Brf required TBP and B" binding required Brf.
The analysis (examples of experiments are shown in Fig. 1b; the
collected data are summarized in Fig. 2)
showed that amino acids 435 to 545 (principally comprising Brf homology
region 2) are essential and sufficient for stable attachment of Brf to
the TBP-DNA complex: B'-DNA complexes with Brf fragments encompassing Brf homology region 2 were readily detected, but the corresponding complex with Brf(1-282) was not (Fig. 1b and 2). Conversely, Brf fragments lacking Brf homology region 2
Brf(1-282), Brf(1-438), and
Brf(164-438)did not form TFIIIB-DNA complexes that were stable to gel
electrophoresis (Fig. 2), although a weak stabilization was noted for
Brf(1-282) at elevated concentrations of B" (data not shown). Further
analysis also showed that the C-terminal 52 amino acids of Brf, which
include Brf homology region 3, were not required for interaction with
TBP or for recruitment of B" into TFIIIB-DNA complexes (Fig. 1b, lanes
d to f and j to l). Photochemical cross-linking experiments (see below)
showed that the amino acid 435-545 segment of Brf, containing homology
region 2, is also necessary and sufficient for B" recruitment to a
stable DNA complex.
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FIG. 2.
Transcription activities and interactions of Brf
fragments. Formation of TFIIIB-DNA and B' (TBP-Brf)-DNA complexes with
the TA-30-B6 DNA probe (Fig. 1a) was analyzed by gel retardation.
Transcription was measured with the SNR6 gene-related
pU6LboxB construct (Fig. 3a). The locations of landmark
features of Brfthe TFIIB-homologous putative Zn-binding domain, the
two TFIIB-related sequence repeats, and the three segments of conserved
sequence among fungal Brfsare indicated at the top, and the N- and
C-terminal Brf deletion mutants are designated at the left. The four
columns at the right summarize the ability of each truncated Brf to
allow formation of B'-DNA and TFIIIB-DNA complexes (nd, not determined;
*, unstable to gel electrophoresis but detected by DNA-protein
photochemical cross-linking) and to allow TFIIIC-independent
transcription directed by the SNR6 (U6) and  TATA boxes
present on plasmid pU6LboxB (+++, 100%; ++, 30 to 80%; +,
5 to 20%; ±, <1 to 4%;  , not reliably detected).
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The preceding experiments specify requirements for formation of
protein-DNA complexes that are sufficiently stable to survive gel
electrophoresis. This is a relatively stringent criterion. A weaker
capacity for interaction with TBP and B" resides in the N-terminal,
TFIIB-related half of Brf and was detected by other means, as shown
below.
Transcription.
The next experiments examined effects of Brf
truncations on TFIIIC-independent transcription of
pU6LboxB, a construct related to the U6 snRNA gene
(SNR6) with two TATA boxes, TATAAATA, in different surrounding sequence; each TATA box generates a pair of
divergent transcripts (Fig. 3a)
(21). As reported previously (21), Brf(165-596)
is able to direct transcription of the pU6L construct,
albeit less efficiently than does intact Brf. Under the conditions of
the analysis, divergent transcription directed by the SNR6
TATA box was considerably diminished (>6-fold), but rightward
transcription directed by the upstream TATA box was much less
affected (Table 1; Fig. 3b [compare
lanes e and h]).
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FIG. 3.
TFIIIC-independent transcription. (a) The
pU6LboxB construct. The start sites and termination sites
of leftward and rightward transcription directed by the SNR6
(U6) TATA box and by the TATA box are indicated. Rightward
transcription initiating to the right of the SNR6 TATA box
can be directed by TFIIIC binding to boxes B and A. (b)
TFIIIC-independent transcription of pU6LboxB with Brf
fragments individually and in combination. Transcripts are identified
at the side [R.M., recovery marker; *, downstream-shifted initiation
with Brf(284-596)], and Brf fragments are indicated above the lanes.
Transcription was performed with 100 fmol of pU6LboxB DNA,
16 fmol of pol III, 100 fmol of TBP, 100 fmol of B", and 300 fmol of
Brf fragment or intact Brf (lanes a to h) or as specified above lanes i
to s. The weaker complementation of Brf(1-320) with Brf(284-596) was
analyzed in lanes q to s with additional TBP (200 fmol) and B" (300 fmol).
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More extensive N-terminal deletions [Brf(284-596), Brf(317-596), and
Brf(435-596)] nearly abolished TFIIIC-independent transcription directed by the U6 TATA box, with only barely detectable levels of
leftward transcription remaining (Fig. 3b, lanes a, b, and r; Table 1).
The transcriptional start site of this residual activity appeared to
shift downstream by ~4 bp (Fig. 3b, lane r). Rightward transcription
directed by the upstream TATA element () was also greatly
diminished, but it was surprising to see retention of some
transcription activity in a Brf fragment entirely lacking its
TFIIB-related component.
C-terminal deletions (Table 1; Fig. 3b) showed transcription only
moderately reduced upon removal of amino acids 545 to 596 (Table 1). To
our surprise, removal of the entire Brf-specific, C-proximal half of
Brf (amino acids 283 to 596) allowed nearly full retention of
transcription directed both from the upstream () and U6 TATA boxes
(Fig. 3b, lane g; Table 1), but this transcription required higher
concentrations of B" than did full-length Brf (data not shown). In
contrast, Brf(1-320) retained very little transcription activity (Table
1; Fig. 3b, lane q). Experiments described below show that Brf(1-320)
also was only weakly active in complementation assays, suggesting that
it may have contained predominantly incorrectly folded material.
Brf(1-438) also was less active than Brf(1-282) (Table 1; Fig. 3b
[compare lanes f and g]), but it is not likely that this was due
solely to a low fraction of correctly folded molecules, because
Brf(1-438) was active in complementation assays described below. It is
also noteworthy that rightward transcription mediated by the TATA box was preferentially lost when amino acids 439 to 596 were removed (Table 1; Fig. 3b, lane f). Combinations of N- and C-terminal deletions
also yielded proteins that either remained or became transcriptionally
inactive: whereas Brf(1-545) was highly active and Brf(164-596)
retained high activity for transcription directed by the TATA box,
Brf(164-545) was essentially inert (lane d), with rightward
transcription mediated by the TATA box only barely detectable (in
the original data; lost in reproduction).
Split Brf.
The availability of many inactive and only
partially active Brf fragments made it possible to test for the
reconstitution of Brf activity by complementation. Examples of
complementation are shown in Fig. 3b (lanes i to s), and the outcome of
a more extensive survey is presented in Table
2. Complementation was assessed by two
criteria: (i) the maximum transcriptional activity achieved with a
combination of two Brf fragments relative to intact Brf; and (ii) the
maximum fold stimulation generated by combining two fragments relative
to the sum of residual activities of each fragment. The first criterion
was satisfied by using relatively high concentrations of all
transcription components and assaying at low ionic strength. The second
criterion is an ideal (near-infinite stimulation) that was achieved for
complementation with Brf(1-282) or Brf(1-438) by lowering the
concentration of transcription factors and/or elevating the ionic
strength. The same reaction conditions were retained for maximal
stimulation and maximal transcription, as long as that allowed an at
least 20-fold increase in one of the pU6LboxB transcripts.
Combining Brf(1-282) with Brf(284-596) generated strong complementation
according to either criterion, with 6- to 9-fold stimulation at
relatively low fragment concentrations (Fig. 3b, lanes j, k, and m) and
16- to 30-fold stimulation at elevated ionic strength (Table 2).
Complementation was also generated when Brf(1-438) and Brf(435-596)
were combined (Fig. 3b, lanes n to p; Table 2), and weak
complementation was detected when Brf(1-320) and Brf(284-596) were
combined (Fig. 3b, lanes q to s). Evidence for complementation of
fragments generated by cleavage at amino acid 164 was principally
provided by combining Brf(1-165) with Brf(164-545) (Table 2).
Brf(164-596) had a relatively high activity of its own (reference
21 and Table 1), and the generally poor activity of
the Brf(1-165) fragment (conceivably reflecting improper refolding of
this poorly expressed protein) generated only equivocal evidence for
complementation of Brf(1-165) and Brf(164-596). The very weak activity
of Brf(164-545) alone (Table 1) made it easier to detect the
complementation.
It also proved possible to remove large internal segments,
reconstituting U6 RNA synthesis with Brf(1-282) and Brf(435-596), for
example (Fig. 3b, lanes j, l, and n). Complementation of Brf(1-282) with Brf(435-545), removing Brf homology regions 1 and 3 by combining a
large internal deletion with a smaller C-terminal deletion, was also
highly effective (Table 2). It was even possible to reconstitute
activity with large polypeptide duplications, combining Brf(1-438) or
Brf(1-320) with Brf(284-596) and combining Brf(1-438) with
Brf(317-596), for example (Table 2; Fig. 3b, lanes q to s). Removing
the N-terminal 163 amino acids (including the putative Zn finger and
the first TFIIB-related repeat) still allowed Brf to be split at amino
acids 435 and 283 (the latter in conjunction with a doubling up of
amino acids 284 to 319), with retention of some activity. All
complementing pairs (Table 2) included Brf homology region 2, pointing
once again to its importance in assembly and function of the
transcription initiation complex.
These results show clearly that Brf can be split at amino acids 283 and
435 and, with retention of partial activity, at amino acid 320. They
imply the existence of at least three independent folding domains in
Brf: amino acids 1 to 282, 283 to 434, and 435 to 596.
Mapping protein and DNA interactions with split Brf.
The
ability to split Brf offers the opportunity to map the interactions of
the resulting fragments with each other and with DNA by photochemical
cross-linking. DNA with the photoactive nucleotide 5-[N'-(p-azidobenzoyl)-3-aminoallyl]dUMP
(ABdUMP) (1, 3) and a radioactive nucleotide inserted
at specific sites of an SNR6 gene-related DNA probe (Fig.
4a) was constructed as
described previously (21). This DNA was designed to generate
unidirectional TFIIIC-independent assembly of TFIIIB-DNA complexes by
virtue of its variant 8-bp TATA box, TGTAAATA, to which
mutant TBPm3 (41) binds in a unique orientation
(47). (Uniqueness of assembly is, of course, crucial for
unambiguous interpretation of cross-linking experiments.)
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FIG. 4.
Mapping the disposition of Brf fragments in
transcription factor complexes by photochemical cross-linking: Brf
split at amino acid 283. (a) DNA probes. The sequence of the
SNR6 gene probe with a TGT mutant TATA box is shown.
Locations at which dTMP was replaced by the photoactive ABdUMP are
circled. The primers used to generate these probes are indicated in
capital letters above and below the sequence. These primers were
extended by incorporating ABdUMP(u) and the indicated radioactive
nucleotide (lowercase letters, underlined) and subsequently extended to
full length. Probes are designated by the location of ABdUMP and by the
photoactive strand (N, nontranscribed; T, transcribed). (b) Analysis of
cross-linking to bp 39/38, 33, and 28 in the nontranscribed
strand. The proteins present with DNA are indicated above the lanes,
and cross-linked proteins are identified at the right. The use of
TBPm3 in conjunction with the TGT-mutated TATA box allows
unidirectional and TFIIIC-independent assembly of TFIIIB on this DNA.
Samples contained 400 fmol of TBPm3, 700 fmol of intact Brf
(designated W), 150 or 300 fmol Brf(1-282) (designated N or
N, respectively), 150 fmol Brf(284-596) (designated C), and 100 fmol
B"(138-594), as indicated. (c to e) Quantitative analysis of
cross-linking efficiencies along the DNA-binding site. The eight probes
indicated between panels c and d were analyzed two, three, or four
times, as indicated in brackets. Cross-linking was quantified,
normalized to the level of the reference protein indicated on the
ordinate of each panel, and averaged. Error bars indicate the average
deviation (for experiments repeated twice) or the standard error of the
mean (for experiments repeated three or four times). Because the
normalization contributed substantially to these variations, rank
orders that were consistently observed are also designated ( between
columns) below the abscissas of panels c and d (for experiments
repeated three or four times only and when not otherwise obvious). (c)
Cross-linking efficiencies of Brf fragments separately and in
combination relative to those of intact Brf in TFIIIB-DNA complexes;
(d) cross-linking efficiencies of B" in TFIIIB-DNA complexes assembled
with Brf fragments separately or in combination, normalized to that of
B" in the TFIIIB-DNA complex with intact Brf; (e) cross-linking
efficiencies of Brf fragments relative to that of intact Brf in
heparin-challenged TFIIIB-DNA complexes reconstituted with Brf(1-282)
and Brf(284-596).
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Complexes of Brf fragments and combinations of fragments with and
without B" were built up on DNA with variously placed ABdUMP residues,
in the presence of excess TBPm3. Sample cross-linking experiments with Brf reconstituted from its 1-282 and 284-596 fragments
are shown in Fig. 4b. Results were analyzed by comparing the
cross-linking efficiencies of the split Brf fragments with that of
full-length Brf within a TFIIIB-DNA complex. The efficiency of B"
cross-linking in complexes with Brf fragments and with intact Brf was
quantified in the same way. In making comparisons in this manner, it is
necessary to bear in mind that the efficiency of cross-linking of
full-length Brf and B" varies between DNA sites, that is, between
probes (Table 3). The N-terminally
truncated B"(137-594) was used for this analysis for technical reasons, but the DNA-binding, transcription, and photochemical cross-linking properties of full-length B" and B"(137-594) are indistinguishable (reference 30 and data not shown). Results of the
analysis for Brf split at amino acid 283 are summarized in Fig. 4c to
e. The following are the salient observations.
(i) Cross-linking of intact Brf to DNA was closely reflected in the
properties of its C-terminal half. When Brf(284-596) was brought to a
TBP-DNA complex by itself, cross-linking was seen between bp 43 and
12 (Fig. 4c) (cross-linking efficiency is expressed relative to that
of intact Brf in the TFIIIB-DNA complex, but it should be noted [Table
3] that intact Brf is relatively inefficiently cross-linked at bp
43, 42, and 32). The level of Brf(284-596) cross-linking did not
significantly change upon separately adding Brf(1-282) or B" (data not
shown). The most striking feature of the cross-linking pattern of
Brf(284-596) by itself (Fig. 4c), with Brf(1-282), or with B" (data not
shown) was the deficit of cross-linking at bp 33 (in the
nontranscribed strand); bp 33 was the preferred site of cross-linking
of full-length Brf in the context of the TFIIIB-DNA complex (Table 3).
Cross-linking of Brf(284-596) at 28N was also markedly
low relative to that of intact Brf (Fig. 4b, middle panel). Forming the
complete TFIIIB-DNA complex with split Brf greatly increased
cross-linking of Brf(284-596) at 33N but had little or
only modest effect elsewhere (in particular, no effect was seen at
28N and cross-linking at 39N reproducibly
decreased). The cross-linking efficiency of full-length Brf was also
lower at 33N in the absence of B" than in its presence,
indicating that efficient cross-linking at this site is also B"
dependent (data not shown).
(ii) Cross-linking of Brf(1-282) was not detectable at any site in the
absence of B" (e.g., Fig. 4b). EMSA also failed to provide evidence
that a complete B' complex formed with Brf split at amino acid 283 (data not shown). The absence of an effect of Brf(1-282) on the
cross-linking of Brf(284-596) in the absence of B" (Fig. 4c) further
indicated a failure to form this complex; Brf(1-282) cross-linked to
DNA at 39/38N, 28N, and
13/12N only when B" was also provided (Fig. 4c and data
not shown), but with comparably low efficiencies (~10% relative to
that of full-length Brf). Addition of B" and Brf(284-596) to form the TFIIIB-DNA complex with split Brf allowed relatively inefficient cross-linking of Brf(1-282) to be detected at 43N,
42T, and 23/22T, as well as enhanced
cross-linking at 39/38N, 28N, and
13N; there was no cross-linking at 33N and
32T. It is noteworthy that the efficiency of Brf(1-282)
cross-linking at 28N preferentially increased and
approached that of full-length Brf. With one exception
(42T), sites of stronger Brf(1-282) cross-linking
relative to that of full-length Brf correlated with weaker
cross-linking of Brf(284-596).
The trend to a neatly partitioned occupancy of DNA sites between the N-
and C-proximal halves of Brf in Fig. 4c and d is partially obscured by
two factors: first, Brf(284-596) formed a stable complex with TBP-DNA
even in the absence of the Brf(1-282) segment and of B"; second,
assembly of Brf(1-282) into the TFIIIB-DNA complex was limiting (data
not shown, but see Fig. 4b) so that some of the cross-linked TFIIIB-DNA
complexes lacked Brf(1-282). Treatment with the polyanion heparin
disrupts partial complexes, and thereby simplifies the analysis, but
was not included in the preceding analysis because it also generates
its own complication: heparin binds to sites within the TFIIIB-DNA
complex that might otherwise be accessible to the photoactive
nucleotide (Fig. 3 of reference 18 and reference 2).
While keeping this complication in mind, it is nevertheless useful to
examine the results of a subsidiary photochemical cross-linking
analysis of heparin-resistant TFIIIB-DNA complexes with Brf split at
amino acid 283 (Fig. 4e): the Brf(284-596) segment accounted for 100%
of the cross-linking of split Brf at 33N and
32T, for 93% of cross-linking at 42T, and
for 37, 20, and 3% of cross-linking at 39/38N,
13/12N, and 28N, respectively.
(iii) B" cross-linked very weakly to DNA in the presence of TBP and in
the absence of Brf or Brf fragments (Fig. 4d). This may reflect the
previously noted direct B"-TBP interaction (37).
(iv) B" was brought into the TFIIIB-DNA complex by either half of Brf
but not with the same disposition relative to DNA (Fig. 4d; note Table
3). In the complex with Brf(1-282), B" was within cross-linking reach
of DNA upstream, at 43N, 42T,
39/38N, and 33N, but not further
downstream, at 32T, 28N, and
23/22T, and only barely at 13/12N. In
the complex with Brf(284-596), B" was cross-linked at all sites: with
nearly full efficiency to full efficiency at 33N and
further downstream, and at somewhat lower efficiency upstream of 33.
Addition of the Brf(1-282) fragment increased the efficiency of B"
cross-linking upstream at 43N, 42T,
39/38N, and, to some extent, 33N but not
further downstream. We conclude that Brf(284-596) holds all of B"
tightly to the TBP-DNA complex and that Brf(1-282) holds only the part
of B" that is in vicinity of DNA upstream of bp 33.
Similar experiments were done with Brf reconstituted from its 1-282 and
435-596 fragments, that is, with the segment from amino acids 283 to
434 missing (Fig. 5). Brf(435-596)
cross-linked poorly at all sites relative to full-length Brf (5 to 20%
relative cross-linking efficiency). The presence of Brf(1-282) and/or
B" had little or no effect on the efficiency of cross-linking of Brf(435-596) (data not shown). Nevertheless, Brf(435-596) by itself proved as competent as Brf(284-596) for recruitment and efficient cross-linking of B". Brf(435-596) increased the cross-linking efficiency of Brf(1-282) in the presence of B", similar to what had
been seen with Brf(284-596) (Fig. 4) but with only one-half of the
quantitative effect (data not shown). Brf(435-545) and Brf(435-596)
were comparably competent in recruitment of B" for cross-linking to the
39/38N probe, but Brf(435-545) was itself cross-linked
poorly (data not shown, but see below).
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FIG. 5.
Analysis of the assembly of DNA complexes with
Brf(1-282) and Brf(435-596) by photochemical cross-linking. The
cross-linking efficiency of Brf(435-596) in the B'-DNA complex is shown
relative to that of intact Brf in the TFIIIB-DNA complex. Recruitment
of B" to DNA by Brf(435-596) and Brf(284-596) is compared and
normalized to the cross-linking efficiencies of B" in TFIIIB-DNA
complexes formed with intact Brf.
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The most straightforward interpretation of these observations is that
amino acids 435 to 545 of Brf, containing Brf homology region 2, serve
as the dominant link between TBP and Brf and also between B" and Brf.
The segment of the C-terminal half of Brf that closely approaches DNA
is contained between amino acids 284 and 434. The N-terminal,
TFIIB-related half of Brf likewise interacts with both TBP and B" but
less stably. Further insight into sites of interaction of each half of
Brf with TBP are provided by the next experiment.
A set of 91 sequence substitutions on the surface of human
TBPm3 (hTBPm3; mutated so as to recognize
TGTAAATA) and an analogous subset of mutations in yeast TBP
were examined recently for ability to promote B'-DNA and TFIIIB-DNA
complex formation with yeast Brf and B" on the SNR6 (U6
gene) TATA box and for ability to promote pol III-directed
SNR6 transcription (6, 12, 40). This analysis defined an extended interaction surface on TBP, where radical sequence
substitutions significantly reduce the affinity of the TBP-DNA complex
for full-length Brf and for the C-terminal half of Brf [e.g.,
Brf(317-596)], implying that most of the affinity for TBP lies in the
C-terminal half of Brf. The Brf interaction epitope of TBP is
positively charged and lies on the top (DNA-distal) surface and side of
that half of TBP that comprises its N-proximal pseudo-repeat sequence.
The Brf interaction surface of TBP is far removed from its
TFIIB-binding surface.
The just-described DNA cross-linking experiments implicate sequences
within amino acids 435 to 545 of Brf in interaction with these epitopes
on TBP. A triple mutation on the top surface of hTBPm3
combining three Brf-binding-defective substitutions (R231E, R235E, and
R239S) was found to nearly abolish TFIIIB-DNA complex formation and
SNR6 transcription (40). The expectation that this construct, hTBPm3(R231E+R235E+R239S), should be
defective in the assembly and photochemical cross-linking of
Brf(435-545) but not of Brf(1-282) was confirmed [compare lanes h and
i in Fig. 6; since photochemical
cross-linking of Brf(435-545) at bp 28 is quite weak, B"
cross-linking provides a simple and more sensitive measure for
Brf(435-545) assembly (compare lanes e to g with a)]. A second triple
mutation in hTBPm3 had also been constructed at three
residues (E284, E286, and L287) that are critical for TFIIB interaction
but have no discernible effect on Brf binding or SNR6
transcription (40). As expected, the E284R-E286R-L287E triple mutation had no effect on the assembly and cross-linking of
Brf(435-545), but Brf(1-282) was now unable to assemble and be
cross-linked to DNA at bp 28 (compare lanes h and j). This result
strongly indicated that the TFIIB-related N-terminal half of Brf
interacts with TBP in a region that overlaps or lies near the
TFIIB-binding domain. (Librizzi and coworkers [32]
previously interpreted a Brf-generated quantitative footprint change to
suggest that Brf might contact TBP in a TFIIB-like manner.) This
interaction between the N-terminal half of Brf and TBP does not appear
to play a significant role in the TFIIIC-independent transcriptional activity of Brf since SNR6 transcription with
hTBPm3(E284R+E286R+L287E) is unimpaired
(40). TFIIIC-dependent transcription on the SUP4 tRNA gene, but not TFIIIB-SUP4 DNA complex formation, was,
however, defective (data not shown).
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FIG. 6.
The N- and C-proximal halves of Brf interact with
opposite ends of TBP: analysis by photochemical cross-linking.
TFIIIB-DNA complexes were formed with 200 fmol of hTBPm3
(WT [wild type]), of the Brf interaction-defective
hTBPm3(R231E+R235E+R239S) (Brf ), or of the
TFIIB interaction-defective hTBPm3(E284R+E286R+L287E)
(IIB), 300 fmol each of Brf(1-282) and Brf(435-545) as
indicated above each lane, 200 fmol of B"(137-594), and 8 fmol of
28N DNA probe. Cross-linked proteins are indicated at the
right. The asterisk marks a fragment of Brf(435-545) that appears to be
competent to assemble TFIIIB-DNA complexes.
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In a recent analysis of the yeast TFIIIB-DNA complex, Colbert et al.
(12) examined the minimum extent of additional DNA that is
required to assemble Brf-TBP-DNA complexes that are stable to gel
electrophoresis. They found extensive additional DNA required downstream of the TATA box but very little, if any, additional DNA
required upstream of the TATA box. In contrast, stable TFIIIB complexes
required a DNA extension upstream of the TATA box and less DNA
downstream than for the Brf-TBP-DNA complex. One might conclude that
these findings imply placement of B" and Brf in a TFIIIB-DNA complex
upstream and downstream, respectively, of the TATA box. The experiments
presented here expose a more feature-rich landscape of Brf and B"
cross-linking to DNA upstream as well as downstream of the TATA box.
Nevertheless, the results of these two analyses are complementary
rather than conflicting, for the following reasons. First, a
determination of minimum DNA size (judged in terms of a particular criterion that is implicit in the specific conditions of a gel electrophoresis assay) does not preclude additional contributions to protein-DNA affinity originating outside that minimum domain. Second, the photochemical cross-linking experiments with
ABdUMP-containing DNA examine close protein proximity, at up to 8 to 9 Å from the pyrimidine ring, rather than direct, in-the-groove
protein-DNA contact. Third, our findings of close Brf proximity to DNA
upstream of the TATA box (Fig. 4 and 5) refer to a TFIIIB-DNA complex. Cross-linking of Brf at bp 33, for example, largely depends on assembly of the TFIIIB-DNA complex (Fig. 4b). This is the result that
one would anticipate if B" binding brought upstream DNA and Brf closer
together, either by further folding the DNA or by generating a
conformational change in Brf.
Interactions with TFIIIC: assembly of transcription factor
complexes and transcription on the SUP4 tRNA gene.
When TFIIIC directs the assembly of the TFIIIB-DNA complex (as it
probably does on all yeast pol III genes, including the U6 snRNA gene,
in vivo [5, 13]), the assembly-initiating interaction
involves Brf instead of TBP (24). The TFIIIC-dependent pathway to transcription places stricter demands on Brf. Of all of the
individual fragments tested, only Brf(1-545) retained a small fraction
of transcriptional activity (~5 to 10% of that of intact Brf), while
the residual activity of Brf(1-282) was only barely detectable (<1%)
and the distribution of transcriptional start sites on the
SUP4 gene was altered (Fig. 7;
compare lane a with lane k). These low residual activities of Brf
fragments made it possible to test with high sensitivity for
reconstitution of activity in TFIIIC-dependent transcription from
combinations of fragments. Brf split at amino acids 283 and 437 was
highly active (Fig. 7; compare lanes a to d and e to h with lanes k and l), generating 45 and 25% of the SUP4 transcription
activity, respectively, of intact Brf. Even the combination of
Brf(1-282) with Brf(435-596) generated readily detectable transcription
activity (10% of the SUP4 transcription activity of intact
Brf) above a low background of TFIIIC-independent transcription (lanes
i and j).
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FIG. 7.
TFIIIC-dependent transcription of the SUP4
gene with Brf split at amino acids 282 and 435. Components (400 fmol of
intact Brf or the indicated fragments, 100 fmol of TBP, 300 fmol of B",
76 fmol of TFIIIC, as indicated, 16 fmol of pol III, and 100 fmol of
the SUP4 gene-containing plasmid pTZ1) are specified above
each lane. SUP4 transcripts and the recovery marker (R.M.)
are identified at the sides.
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The TFIIIC-dependent assembly of DNA-complexes containing Brf fragments
1-282 and 284-596, separately and together, was also examined by gel
retardation, and the internal structures of complexes were probed by
two-dimensional footprinting with MPE-Fe(II) (9). We used
the TA-30 variant of the SUP4 gene for this analysis. In the
TA-30 gene, the AT-rich upstream segment of the SUP4 gene is
replaced by TATAAA centered 30 bp upstream of the
transcriptional start site, surrounded by GC-rich sequence (Fig. 1a).
The TA-30 SUP4 gene restricts initiation of transcription
predominantly to a single site in vitro (in contrast to the wild-type
SUP4 gene [Fig. 7, lane k]) because the TATAAA
box restricts TFIIIB assembly more uniformly to a single upstream
site while still retaining nearly complete TFIIIC dependence for
transcription (19).
TFIIIC-B-DNA complexes formed on the TA-30 gene with Brf split at amino
acid 283 were indistinguishable from complexes formed with intact Brf
by the criterion of MPE-Fe(II) footprinting over the entire length of
DNA covered by the assembled proteins (Fig. 8a). It appeared that all interactions
affecting the disposition of protein around DNA [i.e., Brf-TBP,
Brf-B", and Brf-TFIIIC(
120)] are quite precisely
matched when this split Brf and intact Brf are used to assemble the
TFIIIC-B-DNA complex.
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FIG. 8.
TFIIIC interaction. (a) MPE-Fe(II) footprints of
TFIIIC-B-DNA complexes assembled on TA-30 DNA with intact Brf and with
Brf split at amino acid 283. Reaction mixtures were assembled as
described below and subjected to reaction with MPE-Fe(II) as specified
in Materials and Methods. The resulting material was separated
electrophoretically, and the gel was exposed without drying to a
phosphorimaging screen as shown in panel b. Gel slices corresponding to
the appropriate bands were excised; DNA was extracted, processed,
separated on a sequencing gel as specified in Materials and Methods,
and analyzed on a phosphorimaging detector. The density traces shown
have been normalized at 5' unprotected sequence. Green trace,
TFIIIC-DNA complex; red trace, TFIIIC-TFIIIB-DNA complex assembled with
intact Brf; blue trace, TFIIIC-TFIIIB-DNA complex assembled with
Brf(1-282) and Brf(284-596). (b) EMSA for assembly and stability of
TFIIIC-B'-DNA and TFIIIC-TFIIIB-DNA complexes with Brf(1-282) and
Brf(284-596). Nine femtomoles of TA-30 DNA probe was incubated with 31 fmol of TFIIIC, 200 fmol of TBP, 300 fmol of B", and 200 fmol of intact
Brf (as marked above lanes a to d) or with 31 fmol of TFIIIC, 500 fmol
of TBP, 500 fmol of Brf fragment, and 300 fmol of B" (as indicated
above lanes e to h). Electrophoresis was in Mg2+-containing
gel. The result shown was generated in a footprinting experiment:
DNA-protein mixtures were reacted with MPE-Fe(II) before
electrophoresis, and the exposure shown was made without drying the
gel.
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Each half of Brf was found to interact with TFIIIC, but the evidence
indicated much weaker interactions.
TFIIIC-B[Brf(284-596)]-DNA complexes were
sufficiently stable to be detected as a well-defined band in EMSA (done
in the presence of Mg2+) (Fig. 8b, lane f). A separate
experiment showed that assembly of this complex on the TA-30 gene was
TFIIIC dependent even at the elevated concentrations of components that
were required to drive association with Brf(284-596): the interaction
with the TA-30 DNA probe and its 6-bp TATAAA box was
unstable on the gel in the absence of TFIIIC (data not shown). However,
the MPE-Fe(II) footprint of the TFIIIC-B[Brf(284-596)]-DNA complex,
analyzed by cutting the corresponding band of protein-DNA complex from the gel (Fig. 8b, lane f), was grossly aberrant; for example, it lacked
the footprint segment upstream of the TATAAA box (bp 33 to
42) that is conferred by B" recruitment into the TFIIIC-B-DNA complex
(data not shown). Formation of a TFIIIC-B[Brf(1-282)]-DNA complex was
also detected by EMSA, as judged by a small mobility shift (Fig. 8b,
lane h), and weak TFIIIC-dependent photochemical cross-linking of
Brf(1-282) was detected (data not shown). The observed competence for
transcription, although extremely low, also implied the ability to form
such a complex. The data presented here made very clear that the two
halves of Brf strongly reinforce each other's interactions with
TFIIIC, TBP, B", and DNA.
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DISCUSSION |
Each half of Brf interacts with DNA-bound TBP: the C-proximal half
of Brf interacts with the N-proximal pseudo-repeat lobe of TBP, and the
N-proximal, TFIIB-related half of Brf interacts with the C-proximal
pseudo-repeat lobe of TBP (Fig. 6). A direct interaction between the
C-proximal halves of human and yeast Brf with TBP has been noted
previously, but tests for an interaction with the N-proximal half of
Brf yielded an inconsistent outcome or weak interaction (10, 27,
44). The interaction of the C-proximal half of Brf is the more
stable one, yielding a B'-DNA complex that is detected by gel
electrophoresis (Fig. 1b and 2) and also by photochemical cross-linking
(Fig. 4). Brf also has multiple sites of interaction with B", so that
TFIIIB complexes can be formed by each half of Brf separately (Fig. 1b,
2, and 4). The TBP- and B"-interacting domains of the C-terminal half of Brf both reside within the amino acid 435-545 segment, which contains Brf homology region 2 (Fig. 5 and 6; Table 2). A temperature sensitivity-conferring mutation in this region (L
S at amino acid 462) has been found to yield a Brf protein that is defective in binding
to TBP-DNA (12) either as a result of defective folding or
through loss of a specific protein-protein contact. Brf homology region
2 has a substantial degree of conservation among homologs from a worm
and the human. We anticipate that Brf homology region 2 will also turn
out to be the principal anchor for human Brf on TBP, as has been
suggested on the basis of sequence conservation (34).
Each half of Brf also has some competence to generate transcription,
but the N-terminal half plays the major role in pol III recruitment
(Table 1; Fig. 3b); the relatively high transcriptional activity of
Brf(1-282) and Brf(164-596) in -directed transcription points more
specifically to the importance of amino acids 164 to 282, which
encompass the second TFIIB-related sequence repeat (Fig. 2), in pol III
recruitment. It is likely that the corresponding segment in human Brf
will play a similar role in pol III recruitment and/or initiation of
transcription, since the human pol III RPC39 subunit is homologous to
the yeast pol III rpc34 subunit and likewise interacts with human Brf
(human TFIIIB90) (45).
Some of the truncated Brf proteins differentially retain
TFIIIC-independent transcription activity on our two test promoters. For example, Brf(1-438) was seen preferentially to lose, and
Brf(164-596) was seen preferentially to retain, the capacity for
transcription initiating at the promoter. This is consistent with
two interpretations: (i) formation of a TFIIIB-DNA complex with these
Brf fragments may require interaction with DNA sequence lying outside
the TATA box that is not rate limiting for the transcriptionally more
active full length Brf; and (ii) loss of protein-protein contacts
mediated by the missing Brf segments may place additional requirements on the flexibility of TATA box-flanking DNA for securing required interaction with B" and pol III.
Each half of Brf also interacts with TFIIIC. A small shift of
electrophoretic mobility suggested that Brf(1-282) was brought into a
TFIIIB-DNA complex under the direction of TFIIIC (Fig. 8b), but its
TFIIIC-dependent assembly was only weakly detected by photochemical
cross-linking and its transcriptional activity was extremely low.
Brf(284-596) formed a more stable but structurally aberrant and
transcriptionally inactive TFIIIC-B-DNA assembly (Fig. 8b). It is
surprising to find both halves of Brf required to properly integrate B"
into the preinitiation complex in the presence of TFIIIC, whereas each
half of Brf has the ability to do this in the absence of TFIIIC.
Evidently, the role of TFIIIC as the assembly factor for the TFIIIB-DNA
preinitiation complex (23) is more subtle than one might
have imagined: it brings Brf and TBP to a DNA-binding site but must
also be steered away (by Brf?) from interfering with entry of B" into
the preinitiation complex.
The low transcriptional activity of various N-terminally and
C-terminally deleted Brf fragments on the SNR6 gene
construct and inactivity of these fragments in TFIIIC-dependent
transcription made it possible to test for the retention of
transcription activity when Brf is split at internal sites and when
internal overlaps and gaps are generated. Brf split down the middle (at
amino acid 283) was found to function well for TFIIIC-independent and
TFIIIC-requiring transcription (Table 2; Fig. 7), form a TFIIIC-B-DNA
complex that was indistinguishable from its intact-Brf counterpart in MPE-Fe(II) footprinting (Fig. 8a), and assemble a heparin-stable TFIIIB-DNA complex (Fig. 4f). Brf split at amino acid 437 was also
transcriptionally active (Table 2; Fig. 3 and 7). TFIIIC-independent transcription was seen to be relatively permissive for extensive internal deletions in Brf. However, while Brf homology regions 1 and 3 could be removed, all transcriptionally effective complementing pairs
of Brf fragments included Brf homology region 2 (Table 2; Fig. 3). (We
also encountered some discrepancies [Table 2]. For example, Brf split
between amino acids 317 and 320 [with small duplications] was much
less active than was Brf internally split so as to entirely lack amino
acids 283 to 434 [data not shown]. This and similar inconsistencies
may reflect variable ability to properly fold and/or refold different
overproduced Brf fragments.)
The close correspondence between the properties of intact Brf and Brf
split at amino acid 283 allowed us to examine the disposition of the
two halves of Brf in the TFIIIB-DNA complex. Eight DNA sites, covering
11 bp, were probed in this analysis (Fig. 4c to e). At five of these
sites, cross-linking in the complete TFIIIB-DNA complex was
predominantly to one or the other half of Brf (Fig. 4e). Comparison of
the efficiencies of cross-linking of Brf(284-596) and Brf(435-596)
(Fig. 4c and 5) suggests that proximity of Brf to DNA at bp 33 is
primarily contributed by the nonessential amino acid 284-434 segment
and is not critical to assembly of TFIIIB on DNA.
Further information for constructing a model of the disposition of Brf
in the TFIIIB-DNA complex comes from a recently completed analysis of
TBP mutations that interfere with the TBP-Brf interaction. TBP mutants
interfering with binding of intact Brf also failed to interact with
Brf(317-596) (40), with Brf(352-596) (12), or
with Brf(435-545) (Fig. 6). Conversely, a triple mutation in TBP that
prevents TFIIB binding also prevented cross-linking of Brf(1-282) to a
DNA site at the upstream end of the U6 TATA box in a TFIIIB-DNA complex
assembled with Brf(1-282) and Brf(435-545) (Fig. 6). This result
identifies a site of interaction on the C-proximal lobe of TBP for the
N-proximal TFIIB-related half of Brf and a site on the N-proximal lobe
of TBP for the C-proximal Brf homology domain 2. It is the latter
interaction that contributes the major part of the affinity of Brf for
TBP.
These constraints specify a global model of how Brf assembles into a
TFIIIB-DNA complex. Figure 9 shows the
TBP-DNA complex backbone of this model and identifies six widely
separated reference points that are contacted by Brf. Four points of
close vicinity to DNA, extending from upstream of the TATA box to the
underside and further downstream, are represented by the N atoms (in
yellow) at the tips of ABdUMP side chains (in green) projecting out of the DNA major groove. These sites span more than two and one-half turns
of DNA helix but are brought closer together by the TBP-imposed DNA
bend. Intact Brf cross-links with comparable efficiency to these four
sites in a TFIIIB-DNA complex (Table 3). Two surfaces for
protein-protein contact, one on each lobe of TBP, are represented in
black and gray, respectively.
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FIG. 9.
Model of the disposition of Brf in the TFIIIB-DNA
complex. Two views of the TBP-DNA complex are shown (derived from
reference 35). The existing DNA sequence was changed
to place T at 33N, 32T, 23T,
and 22T, and DNA was also extended at each end with
undistorted B helix (corresponding to SNR6 DNA sequence).
DNA strands are in dark (transcribed) and light (nontranscribed) blue;
core TBP (Arabidopsis thaliana residues 12 to 198) is in
red. The photoactive side chains of ABdUMP at bp 39, 38, 33,
28, 13, and 12 are shown, fully extended and projecting out of
the major groove, in green, with their nitrene nitrogens in yellow. The
TBP residues corresponding to the surface required for stable Brf
binding (40) are shown in black. The corresponding TBP
epitope required for cross-linking of Brf(1-282) and for TFIIB binding
is shown in gray. Gray, dashed, and black arrows point to sites of
proximity and interaction of Brf fragments 1-282, 284-434, and 435-545, respectively. At the right is shown the TFIIB-TBP-DNA complex
(35). The structure comprises amino acids 113 to 316 of
TFIIB (in green), homologous to amino acids 79 to 289 of yeast Brf.
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In order to reach all of these sites, Brf has to intertwine with a
TBP-DNA complex, reaching the underside of the TATA box as well as the
lateral and DNA-distal sides at each end of TBP. Three domains of Brf
make distinctive contacts with these six sites.
Brf(1-282), the TFIIB-homologous segment of Brf, occupies a
TFIIB-related space in the complex. It contacts DNA at the downstream side (13/12N) and under the TBP-imposed bend at
28N but is also within cross-linking reach of the
upstream 39/38N site. The space filled by Brf(1-282)
may be less extended than indicated here because DNA is further bent in
the TFIIIB-DNA complex (4, 15a, 31). The similarity in
disposition of TFIIB and its Brf(1-282) homolog in the pol II and pol
III preinitiation complexes is certainly compatible with a previous
proposal that the relationship between the direction of transcription
and the orientation of TBP is the same for yeast pol II and pol III
(47).
Brf(284-434) contributes the primary DNA proximity of the
C-proximal half of Brf, primarily at 33N but also at
39/38N (Fig. 4f), but this is not essential for Brf
function (Table 2; Fig. 7).
Brf(435-545) is the site of tightest TBP binding, mapped in detail
by Shen et al. (40); the contact surface on TBP is shown in
Fig. 9 in black. This Brf segment is also the site of a tight linkage
to B" (Fig. 4).
The similarity in disposition of TFIIB and of its homologous Brf
segment in space, and the existence of a direct Brf-pol III interaction
(27, 46), raises an interesting question. Why is B"
absolutely required for all yeast pol III transcription, in DNA as well
as in chromatin, in supercoiled as well as linear DNA, in
TFIIIC-dependent as well as TFIIIC-independent transcription, and with
yeast-derived biochemically purified components as well as with
E. coli-synthesized recombinant proteins? Why do Brf and TBP, the B' components of TFIIIB, not suffice for at least some basal
transcriptional activity? It is known that B" drastically changes the
stability of association of the other two components of TFIIIB with
promoters. B" probably also further bends the Brf-TBP-DNA complex. Are
these further structural constraints essential for transcriptional
initiation by pol III? If they are, why is that the case?
It may be helpful to look to bacterial transcription for assessing
these questions and their possible answers. Initiation of transcription
by E. coli RNA polymerase requires successive steps of
promoter attachment (recruitment), isomerization of the closed
promoter-polymerase complex, DNA strand separation around the
transcriptional start site, and promoter clearance. The activities of
bacterial promoters can be limited or activated at each of these steps
(36). We propose that the requirement for B" points to a
role for TFIIIB in transcription by pol III that extends beyond simply
and passively marking the promoter for polymerase attachment.
We thank C. J. Brent and G. A. Letts for assistance
with experiments, H. M. Vu for generous help with construction of
one of the figures, Y. Shen and A. J. Berk for generously
supplying key materials in advance of publication, and C. A. P. Joazeiro for helpful comments on the manuscript.
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Abstract
Introduction
