(Received for publication, February 25, 1997, and in revised form, April 23, 1997)
From the Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique, Saclay, F91191 Gif sur Yvette Cedex, France
TFIIIC-dependent assembly of yeast
TFIIIB on class III genes unmasks a high avidity of TFIIIB for DNA.
TFIIIB contains TATA-binding protein (TBP), TFIIIB90/B", and
TFIIIB70/Brf1, which is homologous to TFIIB. Using limited proteolysis,
we have found that the COOH terminus of TFIIIB70 (residues 510-596)
forms a protease-resistant domain that binds DNA tightly as seen by
Southwestern, DNase I footprinting, and gel shift assays. Consistent
with a role for this DNA binding activity, preinitiation complexes were
formed less efficiently with truncated TFIIIB70 lacking the
COOH-terminal domain and displayed an increased sensitivity to heparin.
B (TFIIIB70 + TBP)·TFIIIC·DNA complexes were also particularly
unstable. In addition, TFIIIB·TFIIIC·DNA complexes containing
truncated TFIIIB70 were impaired in promoting transcription
initiation.
The auxiliary transcription factors required for class III gene
activation have been much investigated, especially in yeast. Essentially all the components of the yeast RNA polymerase III (pol
III)1 transcription system have been
identified and characterized (1, 2). The cascade of interactions
leading to transcription complex formation on some prototypical class
III genes is known in general outline (2-5). Two factors, TFIIIB and
TFIIIC, are required to direct transcription of yeast tRNA genes. The
binding of TFIIIC to the intragenic promoter elements is the primary
step of transcription complex assembly. The TFIIIC·DNA complex
projects its 131 subunit past the transcription start site to
promote the binding of TFIIIB at a fixed distance upstream of the start
site (6-9). TFIIIB can not bind to a tRNA gene by itself. It needs to
be assembled on tDNA, but the resulting TFIIIB·DNA complex is
exceptionally stable and resists disruption by heparin or high salt
concentrations that dissociate TFIIIC (6, 7). Hence, it could be shown that TFIIIB·DNA complexes can direct accurate transcription and reinitiation by RNA polymerase III, in the absence of TFIIIC (7). Therefore, TFIIIB is the central initiation factor responsible for
proper pol III recruitment.
Yeast TFIIIB comprises three components, the TATA-binding protein
(TBP), TFIIIB70/Brf1 homologous to TFIIB, and TFIIIB90/B". TBP was
first shown to participate in pol III transcription in the case of the
yeast SNR6 gene that contains a TATA box at bp 30 (10).
Specific binding of TBP to the TATA box can direct the correct assembly
of TFIIIB in the absence of TFIIIC in vitro (10, 11). TBP
also interacts with DNA on the TATA-less tRNA genes (9), but the
assembly of TFIIIB is then dependent on TFIIIC, via its interaction
with TFIIIB70 (12). TFIIIB70 was shown to interact with the
131
subunit of TFIIIC (13, 14) as well as with TBP (14, 15). The weak
TFIIIB70·TFIIIC·tDNA complex is stabilized after recruitment of TBP
and probably structurally modified, as seen by changes in the
accessibility of both TFIIIB70 and
131 to DNA cross-linking (12).
The B
(TFIIIB70 + TBP)·C·DNA complex is further stabilized and
again modified upon binding of B" that confers the heparin and salt
resistance property typical of B·C·DNA complexes (12, 16). The
recruitment of B" is probably mediated both by B
(17) and the
131
subunit of TFIIIC (18). This succession of interaction steps,
artificially decomposed in vitro, might in fact represent
the in vivo situation, since both genetic (19, 20) and
biochemical (15, 21) evidence suggests that TFIIIB, when not bound to
DNA, is not a stable molecular entity.
The striking stability of the TFIIIB·DNA complex contrasts with the
lack of affinity of TFIIIB or B alone for TATA-less promoters (6, 21,
22). It has been proposed that TFIIIB is convertible between two
states: one binding very tightly to DNA without sequence specificity,
the other hiding the DNA binding domain(s) to avoid random dispersion
of TFIIIB on irrelevant sites (5). Cryptic DNA-binding sites have been
found to exist in general transcription factors like
70,
K, and
32 (23, 24) and RAP30 (25).
Neither intact Escherichia coli
70 nor RAP30
exhibits detectable DNA binding activity, but this property is unmasked
upon removal of the NH2-terminal part of these polypeptides
(23, 25). A masked DNA-binding potential has also been revealed in the
protease-resistant core of TFIIB (26), in the zinc ribbon of TFIIS (27,
28), and in protein p53 (29). To understand how TFIIIB can be locked in
a highly stable protein·DNA complex, we sought for a masked DNA
binding domain in TFIIIB70 using limited proteolysis. We found that the TFIIIB70 COOH terminus folds into a protease-resistant domain that
binds DNA tightly. Deletion of this domain affects the rate of
formation, stability, and function of preinitiation complexes.
Plasmid pET3b(rTFIID) expressing
TBP was a gift from J.-M. Egly (Institut de Génétique et de
Biologie Moléculaire et cellulaire, Strasbourg, France). Plasmid
pSH360 expressing TFIIIB70 was kindly provided by S. Hahn (Fred
Hutchinson Cancer Research Center, Seattle, WA). Plasmid pCC-10,
obtained by cloning of the SalI/XhoI DNA fragment
from YpCC7 (see below) into pET28b (Novagen), encodes a TFIIIB70
derivative from residues 1-509 (rC86) tagged with six histidine
residues and the T7-TAG epitope (Novagen). PCC-Leu3 was constructed by
inserting into pGEM-T (Promega) the
tRNA3Leu gene from residues
150 to
+228 on an NcoI-EcoRI DNA fragment obtained by
polymerase chain reaction.
Oligonucleotide-mediated mutagenesis was performed as described by the
manufacturer, using a Muta-Gene kit (Bio-Rad), on uracil-enriched single-stranded YpCC1 DNA (15). The oligonucleotide
5-GGAGGCAGATATCtaactcgagGCCACAGGTAACAC-3
containing sequences
complementary to PCF4/TDS4/BRF1 DNA (uppercase) and a stop
codon followed by a XhoI restriction site (lowercase) was
used to introduce a stop codon at position +1528 of the PCF4 open reading frame. After sequencing inserts of several transformants, the SacI/SmaI mutagenized DNA fragment was cloned
into the same sites of a multicopy plasmid creating YpCC7. Centromeric
or multicopy plasmids harboring the deleted version of the
PCF4 gene were used to transform the SHy76 haploid strain
(31) containing the PCF4 gene on a plasmid with the
chromosomal copy disrupted. The modified copies of PCF4 were
substituted for wild type PCF4 by plasmid shuffling on
5-fluoroorotic aid plates. The resulting strains isolated at 30 °C
have also been tested for growth at 37 and 16 °C.
Highly
purified RNA polymerase III and affinity-purified TFIIIC were prepared
as described by Huet et al. (52) and Gabrielsen et
al. (53), respectively. TFIIIB activity was reconstituted from the
three components B", TBP, and TFIIIB70. Fraction B" was extracted from
chromatin pellets and partially purified according to the protocols of
Kassavetis et al. (12). Recombinant TBP expressed from
pET3b(rTFIID) was purified by the procedure of Burton et al.
(54). Recombinant histidine-tagged TFIIIB70 or C86 was expressed in
E. coli cells from pSH360 or pCC-10, purified by
chromatography on Ni2+-NTA-agarose (Qiagen) under native
conditions (55). For Southwestern experiments (Figs. 1 and 2) QIAGEN,
rTFIIIB70 was further purified by chromatography on a heparin column.
The protein fraction (0.67 mg of protein) was adsorbed on a 1-ml
heparin column (UGH; Pharmacia Biotech Inc.) equilibrated in buffer A
(25 mM Tris-HCl pH8, 0.12 M KCl, 0.2 mM EDTA, 10 mM
-mercaptoethanol, 15%
glycerol, 1 µM leupeptin, 1 µM pepstatin).
After the column was washed with 8 volumes of buffer A, the proteins
were step-eluted with buffer A containing 0.7 M KCl into
250-µl fractions. Alternatively, rTFIIIB70 was purified on
Ni2+-NTA-agarose under denaturing conditions followed by
Mono S chromatography according to Colbert and Hahn (31). The
recombinant 90-kDa component of TFIIIB (TFIIIB90/B") was purified as
described by Rüth et al. (18).
To purify p10, the 10-kDa protease-resistant domain of rTFIIIB70, fast liquid protein chromatography on a Superdex 75 column (Smart System, Pharmacia) was performed on 5 µg (50 µl) of rTFIIIB70 fraction purified under denaturing conditions. Polypeptides were eluted with a buffer containing 50 mM Tris-HCl, pH 8, 500 mM KCl, and 10% glycerol. About 50 fractions (43 µl) were collected and analyzed by SDS-PAGE and silver staining. Fraction 17 (27.5 µg/ml) was found to contain most of the 10-kDa polypeptide.
Limited proteolysis of heparin-purified rTFIIIB70 was performed according to the procedure of Cleveland et al. (56) with the following modifications. After SDS-PAGE (13%), the full-length rTFIIIB70 polypeptide was located by Coomassie Blue staining. The gel was washed with water, and the protein band was excised and loaded on a SDS-polyacrylamide gel (13%) together with proteinase K or protease V8 as described except that 2 mM EDTA and 5% glycerol were added in the upper gel (57). The polypeptides generated during the co-migration of rTFIIIB70 with the proteases were transferred onto nitrocellulose and revealed with antibodies directed to TFIIIB70 or probed with labeled tDNA.
Amino-terminal Sequence AnalysisHigh pressure liquid chromatography was performed using a chromatograph 130-A (Applied Biosystems). Chemicals for buffer preparation were purchased from Pierce (trifluoroacetic acid), J. T. Baker Inc. (acetonitrile), and Merck (isopropyl alcohol). A Millipore system supplied high pressure liquid chromatography quality water. Chromatographic separations were performed at 35 °C. Recombinant TFIIIB70 fraction purified under denaturing conditions (10 µg) was diluted with water up to 450 µl and then applied to an Aquapore RP300 column (2.1 × 30 mm; Brownlee Labs). The column was developed in solvent A with a linear gradient from 25 to 50% of solvent B for 30 min at a flow rate of 200 µl/min (solvent A was 0.1% trifluoroacetic acid in water, and solvent B was 0.075% trifluoroacetic acid in 40% acetonitrile, 40% isopropyl alcohol, and 20% water). Peaks of absorbance at 214 nm were collected and analyzed for protein content by SDS-PAGE. For amino-terminal sequence analysis, the fraction containing the purified 10-kDa fragment of rTFIIIB70 was partially dried in an evaporator (Speed-Vac, Savant) and spotted onto a Polybrene-pretreated filter disc. Sequence analysis was performed on an Applied Biosystems model 477-A liquid phase sequenator interfaced with a model 120-A on-line phenylthiohydantoin-amino acid analyzer.
Interaction of rTFIIIB70 with TBP or DNAFar Western analysis was performed using in vitro synthesized 35S-labeled TBP as described previously (15). For Southwestern analysis, rTFIIIB70 fractions were subjected to SDS-PAGE and blotted onto nitrocellulose. The filters were then incubated for 1 h at 4 °C in buffer B (20 mM Hepes-KOH, pH 7.9, 0.1 mM EDTA, 5% glycerol, 1 mM dithiothreitol, 5 mM MgCl2) containing 100 mM KCl, in the presence of 100,000 cpm/ml of a 32P-labeled DNA fragment harboring the SUP4 tRNATyr gene (270-bp BamHI-BamHI fragment from plasmid pTZ1; Ref. 6). The filters were washed for 10 min at 4 °C, three times with buffer B containing 100 mM KCl, and twice with the same buffer without glycerol. Labeled polypeptides were revealed by autoradiography.
In Vitro Transcription AssaysPlasmid DNAs used for
in vitro transcription were the following: pRS316-SUP4,
containing the yeast SUP4 tRNA gene (a gift from S. Shaaban); pUC-Glu (58), harboring the
tRNA3Glu gene; pY41 and pY44 (59),
bearing the tRNA2Arg and
tRNA3Ser genes, respectively; pB6 (60),
bearing the SNR6 gene; and pGE2-WT, bearing the
tRNA3Leu gene (61). Transcription
reactions (40 µl) containing 20 mM HEPES-KOH, pH 7.9, 0.1 mM EDTA, 100 mM KCl, 7 units of RNasin (40,000 units/ml; Promega), 0.1 mM dithiothreitol, 5 mM
MgCl2, 10% glycerol, a 0.6 mM concentration
each of ATP, CTP, and GTP, and 0.03 mM
[-32P]UTP (10 µCi) were programmed with 125 ng of
plasmid DNA, 50 ng of affinity-purified TFIIIC (except for
SNR6 transcription), 50 ng of RNA polymerase III, 40 ng of
rTBP, 400 ng of B", and rTFIIIB70 (150 ng) or r
C86 (175 ng) and
incubated for 45 min at 25 °C. RNA products were recovered and
quantitated as described (15). For synthesis of the 17-mer (6),
pRS316-SUP4 DNA (125 ng) was preincubated in 35 µl of transcription
buffer for 20 min at 22 °C with 50 ng of TFIIIC, 40 ng of rTBP, 400 ng of B", and rTFIIIB70 (50 ng) or r
C86 (140 ng). Pol III (50 ng),
ATP and CTP (0.5 mM each), and [
-32P]UTP
(5 µM; 20 µCi) were then added (5 µl), and incubation
continued for up to 20 min as indicated. The reaction was stopped by
adding 1% SDS (final concentration), and RNA products were purified by phenol extraction and double ethanol precipitation and separated on
15% polyacrylamide gels containing 7 M urea. Transcripts
were quantitated by a PhosphorImager with ImageQuant software
(Molecular Dynamics).
For
p10·DNA complex analysis by DNase I footprinting, 5 fmol (12,000 cpm)
of a 5-end 32P-labeled DNA fragment carrying the yeast
SUP4 gene (212-bp BamHI-SmaI fragment
from the pTZ1 plasmid; Ref. 6) were incubated for 30 min at 22 °C in
20 µl of binding buffer containing 20 mM Tris-HCl, pH 8, 0.5 mM EDTA, pH 8, 100 mM KCl, 10% glycerol
with varying concentrations of p10 polypeptide purified by gel
filtration. The DNase I treatment was initiated by the addition of 5 mM MgCl2, 0.5 mM CaCl2,
and 6 ng of DNase I (BDH 39101 RNase-free). After 30 s at
22 °C, reactions were stopped with 10 mM EDTA, and the cleavage products were purified by two phenol extractions and ethanol
precipitations, separated on 8% polyacrylamide gels containing 8 M urea, and revealed by autoradiography.
Preinitiation complexes were assembled on a 284-bp
32P-labeled XbaI-EcoRI DNA fragment
from the pTZ1 plasmid carrying the yeast SUP4 gene. A
mixture (10 µl) of rTFIIIB90 (30 ng), rTBP (30 ng), TFIIIC (50 ng),
and rTFIIIB70 or rC86 (40-400 ng, as indicated) preincubated for 10 min at 22 °C was then incubated for 20 min at 22 °C in binding
buffer (20-µl final volume) with 2 fmol (~3000 cpm) of
32P-labeled template and 150 ng of pBR322. With the
tRNA3Leu gene as a template,
TFIIIB
·TFIIIC·DNA complexes were formed in the absence of
rTFIIIB90, using a 358-bp 32P-labeled
NcoI-EcoRI DNA fragment from the pCC-Leu3
plasmid. When indicated, variable concentrations of heparin (Sigma,
H-2149) were added to the reaction mixtures and incubated for 5 min at 22 °C. Complexes were analyzed by nondenaturing gel electrophoresis at 4 °C in 4% polyacrylamide gels (53).
For two-dimensional footprinting assays, B·C·DNA complexes were
formed as described above with the 5
-end labeled
tRNA3Leu gene as a template. Complexes
were then treated with DNase I (6 ng) for 1.5 min at 22 °C, and
reactions were stopped with 20 mM EDTA. Complexes were
separated from free DNA by nondenaturing gel electrophoresis in 4%
polyacrylamide gels and located by autoradiography. Retarded and
nonretarded DNAs were excised from the native gel, passively eluted in
10 mM Tris-HCl, pH 8, 1 mM EDTA, precipitated with ethanol, analyzed on a 10% polyacrylamide gel containing 8 M urea, and revealed by autoradiography.
As shown in Fig. 1, preparations of recombinant, histidine-tagged TFIIIB70 purified under denaturing (lane 1) or native (lane 2) conditions contained polypeptides ranging from 10 to 70 kDa. Most of these polypeptides were recognized by antibodies directed to TFIIIB70 (lanes 3 and 4) or to the carboxyl-terminal histidine stretch (lanes 7 and 8). We took advantage of this partial proteolysis of TFIIIB70 to investigate the TBP and DNA binding properties of TFIIIB70 domains. In previous work, we found that TBP interacts with the carboxyl-terminal extension (CTE) of TFIIIB70 (13). Consistent with this result, we found that TBP interacted with carboxyl-terminal fragments of TFIIIB70 larger than 30 kDa (lanes 5 and 6). The same rTFIIIB70 polypeptides were blotted and probed in parallel with a 32P-labeled DNA fragment bearing the SUP4 tRNA gene. Wild type TFIIIB70 and most of the large polypeptide fragments did not bind tDNA. However, both preparations of rTFIIIB70 contained a 10-kDa fragment that bound the tDNA probe. Note that the 30-kDa subdomain of rTFIIIB70 that bound TBP and corresponded to the carboxyl-terminal part of the CTE (lane 10) also bound to tDNA. The recombinant fraction enriched in the 10-kDa fragment (lane 1) also interacted with the tDNA probe in gel shift assays to give one retarded band of complex (data not shown). The same retarded band was obtained with the purified 10-kDa fragment (see below). These results confirmed that the entire TFIIIB70 protein is not able to bind DNA by itself and suggested that TFIIIB70 contains a cryptic DNA binding domain.
To ascertain that the 10-kDa DNA binding polypeptide corresponded to a domain of rTFIIIB70 and was not a contaminant E. coli protein, the full-length TFIIIB70 protein was gel-purified and subjected to limited proteolysis (Fig. 2). Proteolytic fragments derived from rTFIIIB70 were revealed with antibodies or probed with labeled tDNA. As shown in Fig. 2, proteinase K or protease V8 generated fragments of TFIIIB70 of various molecular weights that for the most part did not bind DNA. However, two or three polypeptides of low molecular weight were revealed by the tDNA probe (Fig. 2). Cleavage by both proteases gave rise to a DNA binding domain of about 10 kDa that probably corresponded to the recombinant protease-resistant 10-kDa fragment. The 10-kDa polypeptide (p10) from the recombinant TFIIIB70 preparation was purified by gel filtration and used in subsequent work.
TFIIIB-DNA interaction is exceptionally stable to high electrolyte concentrations (6, 7). We therefore explored the effects of salt concentration on p10·DNA complex formation or stability, in Southwestern experiments. The purified p10 polypeptide was subjected to SDS-PAGE and transferred onto a membrane. Filters were incubated in binding buffer containing labeled tDNA and different concentrations of KCl. The p10 polypeptide bound DNA optimally in the presence of 0.1 or 0.2 M KCl. Complex formation was only partially reduced in the presence of 0.4 M KCl but totally inhibited at 1 M salt. Once formed, the complexes were stable for up to 1 h in 0.4 M KCl but were destroyed by 0.6 M salt, heparin (0.1 mg/ml), or proteinase K (data not shown). Thus, the 10-kDa domain of rTFIIIB70 was capable of forming tightly bound, high salt-resistant complexes.
The p10·tDNA complex was further analyzed by DNase I footprinting.
The DNase I digestion patterns of the transcribed strand of the
SUP4 gene in the presence of increasing amounts of bound p10
polypeptide is shown Fig. 3. p10 did not bind to DNA in
a sequence-specific way, since no specific footprint could be precisely observed. Interestingly, protein-bound DNA showed a series of enhanced
DNase I cleavage sites, regularly spaced every 15-20 bp (indicated
with black dots, for example at bp 25,
9, and +14). Many
of these cleavage sites were not present in the absence of p10. This
was the case at bp
25, just 3
of one of the AT-rich regions present
in the upstream sequence of the SUP4 gene (indicated in Fig.
3 as TATA) or at bp +37/+38. This suggests that p10
molecules bound in a regular pattern along the DNA and possibly induced a structural change in the DNA favoring DNase I attack.
To map the protease cleavage site, the p10 polypeptide was purified by
high pressure liquid chromatography and subjected to NH2-terminal sequence analysis. Based on sixteen
consecutive residues that could be clearly determined, p10 was a well
defined COOH-terminal fragment starting at A510 and encompassing the
last 87 amino acids of TFIIIB70. The precise proteolytic cleavage of
TFIIIB70 COOH-terminal domain suggests a very stable structural domain.
The carboxyl-terminal end of rTFIIIB70 from residue 510 to 596 was
expressed in E. coli, purified under native conditions on a
nickel-agarose column, and tested in Southwestern or gel retardation
assays for tDNA binding. All results described with p10 polypeptide
purified by gel filtration were reproducibly obtained with recombinant
p10 (data not shown). p10 encompasses one of three regions strongly
conserved in the CTE of TFIIIB70 from different yeast species (Fig.
4), starting at the end of region II, as defined by Khoo
et al. (14). As seen in Fig. 4, sequence similarities could
also be found with a human equivalent of TFIIIB70 (30) and with a
putative protein of Caenorhabditis elegans. The
CTE-(510-596) corresponds to a region of low sequence similarity with
HMG2 in human TFIIIB90 (30). Since sequence conservation was a sign of
functional significance, we explored the ability of C86 (truncated
TFIIIB70 deprived of the CTE-(510-596) sequence) to assemble into
stable preinitiation complexes and to direct transcription
initiation.
Transcriptional Activity and DNA Binding Properties of the Deleted Form of TFIIIB70
Previous work has shown that a yeast strain
expressing a derivative of TFIIIB70 deleted of 50 residues at its
carboxyl-terminal end was thermosensitive and cryosensitive, whereas a
larger deletion of 100 residues was lethal (31). To study the phenotype
of strains harboring TFIIIB70 truncated from residue 510 to 596 (thereafter named C86), we constructed this derivative by
site-directed mutagenesis as described under "Experimental
Procedures." Centromeric or multicopy plasmids harboring the
C86
mutant copy of the TFIIIB70 gene were tested for their ability to
functionally replace, at different temperatures, a chromosomally
disrupted copy of the gene. We found that the
C86 deletion was
thermosensitive at 37 °C and cryosensitive at 16 °C when the
mutant gene was on a multicopy plasmid but lethal at 30 °C when
expressed from a centromeric plasmid. Therefore, the carboxyl-terminal
end of TFIIIB70 was essential for cell viability when
C86 was
expressed from a low copy number plasmid from its own promoter.
The C86-deleted form of TFIIIB70 was tested in vitro for
its ability to replace full-length TFIIIB70 for specific transcription of various pol III genes. Fig. 5 shows a comparison of
the transcriptional efficiencies of rTFIIIB70 and r
C86. Several pol
III genes were transcribed in a reconstituted transcription system, in
the presence of rTFIIIB70 or r
C86 at about the same concentration.
The deleted form of TFIIIB70 showed reduced levels of transcription
over wild type with all of the templates tested. The difference in
transcription efficiency varied from 5-fold for the
tRNA3Lue gene to 8-fold for the SUP4
or SNR6 genes, and no transcription at all could be detected
for the tRNA3Ser or
tRNA2Arg genes. The faint transcript seen with
the tRNA3Glu gene was not quantified. This
variable response of the different templates to truncated TFIIIB70
suggested a critical involvement of their upstream TFIIIB-binding
sequences. The SUP4 tRNA gene was chosen to further
investigate the reasons for the low transcription efficiency of
r
C86, since transcription of this gene has been extensively studied
in vitro (3). First, varying the concentration of r
C86
over a large range (20-400 ng) did not restore the level of
transcription observed with 50 ng of rTFIIIB70 (a maximum of 20% of
wild type transcription level was reached); in competition experiments,
the addition of an excess of r
C86 (140 ng) over rTFIIIB70 (50 ng)
did not inhibit transcription; however, in preemption experiments,
where r
C86 was first preincubated with all of the factors, the
addition of rTFIIIB70 could not restore normal transcription efficiency, although in the absence of B", rTFIIIB70 was dominant over
r
C86 to form productive B
·C·DNA complexes (data not shown). These results suggested that r
C86 was poorly assembled in
preinitiation complexes and that complexes containing the deleted form
of TFIIIB70 were somehow deficient in transcription initiation. The
following experiments were designed to confirm these conclusions.
The ability of rC86 to direct transcription initiation was examined
after preassembly of TFIIIB. TFIIIC·DNA complexes, formed on a
labeled SUP4 tDNA gene, were preincubated for 20 min with rTBP, rB", and increasing amounts of rTFIIIB70 or r
C86, before the
addition of pol III and nucleotides. Ternary complex formation, monitored by the synthesis of a 17-mer nascent RNA in the absence of
GTP, was initiated less efficiently with the deleted r
C86 protein
than by the wild type (Fig. 6). Increased amounts of pol III or preincubation of pol III with preinitiation complexes before the
addition of the three nucleotides did not restore wild type synthesis
of the 17-mer product (data not shown). These results suggest that the
defect in transcription initiation caused by r
C86 was not due to a
lower affinity of pol III for the preinitiation complexes that could be
compensated by increasing pol III concentration but that it may
correspond to differences in the number, stability, and/or conformation
of preinitiation complexes.
To test this hypothesis, TFIIIB·TFIIIC·tDNA complexes were
separated from free SUP4 tDNA by native electrophoresis and
visualized by autoradiography. As shown in Fig.
7A, the B·C·DNA complex was fully formed
in the presence of 80 ng of rTFIIIB70 (lane 4), whereas 420 ng of rC86 were necessary to obtain a similar result (lane 12). Detectable complex formation with r
C86 required 240 ng of protein (lane 11). That concentration of r
C86 stabilized
all C·DNA complexes, as seen by the disappearance of free DNA, but only ~50% of the complexes were fully shifted, while an intermediate complex, migrating slightly slower than C·DNA complexes, was formed (lane 11). Since we had verified by silver-stained SDS-PAGE
and by Western blot analysis that the protein content of both
recombinant fractions was similar (data not shown), r
C86 was clearly
less efficient than wild type TFIIIB70 in assembling B·C·DNA
complexes on the SUP4 tDNA gene. This result accounted in
part for the low levels of transcription initiation observed previously
and was also consistent with the in vivo phenotype of mutant
yeast strains that could survive only when
C86 was overexpressed.
Nevertheless, we noted previously that 400 ng of r
C86, corresponding
to 100% of B·C·DNA complexes formed on a SUP4 template
(lane 12), did not restore wild type levels of
transcription. Furthermore, even if the tRNA3Ser
tDNA gene was not detectably transcribed in transcription mixtures containing r
C86 (see Fig. 5), a B·C·DNA complex could be formed with r
C86 on this template (data not shown). These observations indicated that B·C·DNA complexes assembled in the presence of r
C86 were not functionally identical to the ones formed with rTFIIIB70.
Formation and heparin resistance of
preinitiation complexes containing full-length or truncated rTFIIIB70.
A, formation of TFIIIB·TFIIIC·SUP4 DNA
complexes. Factor·DNA complexes were formed as described under
"Experimental Procedures" with TFIIIC alone (lane 2), or
TFIIIC + rTBP + rTFIIIB90 and varying amounts of rTFIIIB70 (40, 80, 120, 240, and 400 ng, respectively, in lanes 3-7) or
rC86 (43, 87, 131, 246, and 422 ng, in lanes 8-13).
Complexes were analyzed by electrophoresis and autoradiography. The
free DNA probe is in lane 1. B,
heparin-resistance of TFIIIB·TFIIIC·SUP4 DNA complexes.
Factor·DNA complexes were formed as described above with rTFIIIB70
(400 ng) or r
C86 (523 ng) and then incubated for 5 min at 22 °C with varying
concentrations of heparin and analyzed by electrophoresis. Lane
1, DNA probe; lane 2, TFIIIC·DNA complex; lanes
3 and 8, control B·C·DNA complexes. B·C·DNA
complexes treated with heparin at 2 µg/ml (lanes 4 and
9); 4 µg/ml (lanes 5 and 10); 10 µg/ml (lanes 6 and 11); 20 µg/ml (lanes
7 and 12) are shown. C, heparin resistance
of B
·TFIIIC·Leu3 tDNA complexes. Factor·DNA complexes were
formed with TFIIIC alone (lane 2), TFIIIC + rTBP + rTFIIIB70
(400 ng, lane 3), or r
C86 (523 ng, lane 8) as
described under "Experimental Procedures." B
·C·DNA complexes were treated with heparin at 1 µg/ml (lanes 4 and
9), 2 µg/ml (lanes 5 and 10), 4 µg/ml (lanes 6 and 11), or 10 µg/ml
(lanes 7 and 12). The migration of the different
factor·DNA complexes is indicated.
The properties of TFIIIB·TFIIIC·tDNA complexes were therefore
further investigated. Complexes assembled on a labeled SUP4 tDNA gene in the presence of TFIIIC, rTBP, rB", and rTFIIIB70 (400 ng)
or rC86 (525 ng) were incubated for 5 min with increasing amounts of
heparin. It was shown previously that incubation of a B·C·DNA
complex with heparin stripped off TFIIIC from DNA and resulted in the
formation of stable TFIIIB·tDNA complexes (6, 7). As shown in Fig.
7B, heparin treatment of B·C·DNA complexes assembled in
the presence of r
C86 resulted in the formation of lower amounts of
TFIIIB·tDNA complexes than with the wild type protein and in release
of free DNA (compare lanes 6 and 7 to lanes 11 and 12). Quantification showed that the ratio of
B·C·DNA complexes (lane 3 or 8; no heparin)
to B·DNA complexes (lane 7 or 12; 20 µg/ml
heparin) was 1.2 and 4.4 for the wild type and truncated protein,
respectively. Four independent experiments gave similar results:
B·C·DNA complexes containing r
C86 gave rise, when incubated with
heparin (20 µg/ml), to ~3.5-fold fewer TFIIIB·tDNA complexes than
wild type TFIIIB70. These results indicated that B·C·DNA complexes
were less stable with r
C86 than with TFIIIB70 or were more sensitive
to disruption by polyelectrolytes. B·C·DNA complexes may also be
structurally different because, when subjected to mild proteolysis, the
complexes formed with r
C86 generated a protein·DNA electrophoretic
band pattern noticeably different from that obtained with rTFIIIB70
(data not shown).
We also explored the stability of the intermediate complex
B·C·DNA, formed in the absence of B" (Fig. 7C; for this
experiment, we used Leu3 tDNA, since B
·C·DNA complexes are well
resolved from C·DNA complexes). This type of complex is much less
resistant to heparin than the B·DNA complex (12). Interestingly,
heparin treatment of B
·C·DNA complexes containing r
C86
generated a new complex, C
, migrating almost like TFIIIC·DNA
complexes and similar to the intermediate complex observed above (Fig.
7A, lane 11). The C
complex was resistant to
concentrations of heparin that totally disrupted TFIIIC·DNA complexes
(1 µg/ml). The C
complex was also observed when B·C·DNA
complexes were treated with 2 or 4 µg/ml heparin (see Fig.
7B, lanes 9 and 10). Its faster
migration rate may reflect the loss of polypeptides from B
·C·DNA
complexes or, more probably, a large conformational change like the
loss of DNA bending characteristic of B·C·DNA complexes (32).
We finally investigated whether DNA protection was altered in
B·C·DNA complexes formed with r
C86 on the
tRNA3Leu gene. Preformed B
·C·DNA complexes
were treated with DNase I and separated from free DNA by native
polyacrylamide electrophoresis (Fig. 8A)
before analysis of the DNase I cleavage pattern on a sequencing gel.
The DNase I digestion patterns of the transcribed strand of the
tRNA3Leu gene in free DNA or in B
·C·DNA
complexes is shown in Fig. 8B. The footprint of TFIIIC over
the A and B blocks was clearly observed. B
extended DNase protection
from bp +1 to bp
35 much as described for SUP4 tDNA (12,
21). Two differences were seen with r
C86. First, although preformed
complexes were separated from free DNA before DNA analysis, the
footprint over the +1/
38 region was incomplete, which suggested an
instability of B
·C·DNA complex during DNase treatment. In
addition, the size of the footprint was slightly reduced on both sides.
In particular, there was an enhanced cleavage site at
35 that was
absent in free DNA and in complexes containing TFIIIB70. This footprint
analysis, therefore, confirmed the alteration of B
-DNA
interaction.
TFIIIB70 or Brf1 is a pivotal component of TFIIIB, since it interacts with multiple components of the transcription complex including TFIIIC, TBP, and the C34 subunit of RNA polymerase III (13-15, 33). The amino-proximal half of TFIIIB70 is structurally related to TFIIB, with a putative Zn2+-binding sequence, followed by two imperfect repeats (19, 31, 34). The proteolytically stable core TFIIB, which consists of the two repeats, interacts with TBP and with DNA upstream and downstream of the TATA box (26, 35-39). DNA bending by TBP is critical for fitting TFIIB in the complex. Based on its homology with TFIIB, the amino-terminal half of TFIIIB70 is probably inserted similarly in the TFIIIB·DNA complex. However, a major structural difference is the existence in TFIIIB70 of a CTE that almost doubles the size of the polypeptide compared with TFIIB. Unexpectedly, it is the CTE that most strongly interacts with TBP (13, 14). Here we show that the COOH-terminal region of TFIIIB70 (CTE-(510-596)) forms a protease-resistant domain that has the property to bind DNA tightly, in contrast to full-length TFIIIB70. This domain is important to form and stabilize TFIIIB·DNA complexes and to direct efficient initiation of transcription.
The existence of a DNA binding domain located at the COOH terminus of TFIIIB70 was revealed by limited proteolysis, a method often used to delineate functional regions in proteins (26, 29, 35). The precise proteolytic cleavage occurring naturally in E. coli cells is strongly suggestive of a tightly folded structural domain. The interaction of the p10 polypeptide with DNA displayed several interesting properties: it was remarkably resistant to electrolytes (0.4 M salt), apparently nonspecific (since binding was detected by footprinting all over the SUP4 tRNA gene), and possibly caused a distortion of the DNA backbone as suggested by the regularly spaced DNase I enhanced cleavage sites. Alternatively, these enhanced cleavage sites may also simply correspond to hypersensitive sites between closely spaced p10 molecules.
The hypothesis that CTE-(510-596) contributes by its DNA-binding
property to TFIIIB·DNA complex formation and stability was supported
by several observations. First, the deletion of residues 510-596
impaired the formation of B·C·DNA complexes. Five times as much
rC86 was needed for full complex formation on SUP4 tDNA as compared with intact TFIIIB70. Second, B·C·DNA complexes
containing the truncated form of TFIIIB70 were less resistant to
heparin than wild type complexes. Third, B
·C·DNA complexes formed
with r
C86 were particularly unstable. This instability was observed in two-dimensional footprinting experiments where isolated complexes displayed only partial protection of the upstream region; it was also
apparent in transcription-competition experiments where intact rTFIIIB70 was dominant over r
C86 for B
·C·DNA complex formation (data not shown). Interestingly, heparin treatment of B
·C·DNA complexes generated a new form of complex (called C
, since it migrates
just slightly slower than C·DNA complexes) that was not obtained with
wild type TFIIIB70. C
complex was also observed as an intermediate
during B·C·DNA complex formation with limiting levels of r
C86.
There is the possibility that heparin treatment induced the loss of
r
C86 from the B
·C·DNA complex that may explain the migration
shift. Alternatively, the large down-shift of C
·DNA complexes
compared with B
·C·DNA could reflect an important conformational change of the complex. TFIIIB was shown to bend DNA (32), and B
contributes to this bending (40). A TBP-induced bend in the TATA-less
upstream region could require TFIIIB70 to be stabilized. Complete or
partial dissociation of r
C86 by heparin may then cause a loss of DNA
bending and increase the migration rate of the complex. Whatever the
cause, the formation of the C
complex further underscores the role of
the COOH terminus of TFIIIB70 in complex stability. One could imagine
that the COOH-terminal DNA binding domain of TFIIIB70 is unmasked
during the recruitment of TBP and holds the B
·DNA complex in place
(note that p10 by itself does not bind TBP; see Fig. 1).
Transcription studies suggest that the role of the CTE-(510-596)
domain may not be restricted to a firmer anchoring of TFIIIB70 on the
DNA. The addition of an excess of rC86, leading to full B·C·DNA
preinitiation complex formation, in fact, did not restore wild type
transcription efficiency. In addition, different templates were
transcribed with largely varying efficiencies in the presence of
r
C86. Hence, the tRNA3Ser gene could form a
B·C·DNA complex of apparently normal electrophoretic migration with
r
C86 but was very poorly transcribed. Transcription initiation,
monitored by synthesis of a short nascent RNA on SUP4 DNA
remained impaired in the presence of an excess of RNA polymerase III
(data not shown). Thus, B·C·DNA complexes containing r
C86 were
deficient at some stage of transcription initiation. If p10 polypeptide
indeed distorts the DNA backbone, there is the possibility that the
COOH terminus of TFIIIB70 facilitates DNA melting by RNA polymerase.
Attempts to reconstitute TFIIIB70 transcriptional activity by
supplementing r
C86 with excess p10 were only partially successful
(2-3-fold stimulation; data not shown).
Considering the striking structural and functional homology of TFIIB
and TFIIIB70, the adjunction of the CTE to the NH2-terminal TFIIB-like domain is intriguing. The COOH-terminal extension of TFIIIB70 could possibly play two types of function. One function would
be to orient TBP·DNA complexes irreversibly toward pol III transcription. In this respect, the CTE can be considered equivalent to
a class III TBP-associated factor. In addition, the CTE could have a
basic function played by a pol II factor distinct from TFIIB. The best
candidate for a functional homologue would be RAP30, the small subunit
of TFIIF(RAP30/74). TFIIF interacts with RNA polymerase II and
cooperates with TFIIB to recruit the enzyme into the preinitiation
complex (41). RAP30 is critical in this process, since it was shown to
bind both TFIIB and RNA polymerase II (42-44) and to be sufficient for
specific recruitment of the enzyme to a TBP·TFIIB·promoter complex
(45-47). The CTE of TFIIIB70 provides a similar link between TBP, the
TFIIB-like region of TFIIIB70, and the C34 subunit of pol III (13-15,
33). In addition, like the CTE of TFIIIB70, RAP30 has a cryptic DNA
binding domain of similar size (~80 amino acids) located at the COOH
terminus (25, 48). These domains, CTE-(510-596) and RAP-(162-249), bind DNA nonspecifically, which is not unexpected, since, in both cases, sequence specificity is provided by other components, and their
deletion impairs transcription initiation (Ref. 48 and this work).
RAP30 is homologous to transcription factor 70 (49, 50).
It has been proposed that the DNA binding domain of RAP30 is related to
an evolutionary conserved DNA binding domain of members of the
70 family of
factors (51). A structural and
mutational comparison of the DNA binding domains of RAP30 and TFIIIB70
would shed light on the evolution of the eucaryotic transcription
machineries.
We thank Jean-Christophe Andrau and Eric Deprez for help, Steven Hahn for the gift of plasmid, strain, and anti-BRF1 serum, and Jean-Marc Egly for providing the pET3b/rTFIIDY plasmid. We thank Giorgio Dieci, Olivier Lefebvre, and Peter Geiduschek for helpful discussions, Françoise Bouet for peptide sequences, and Carl Mann for improving the manuscript.