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Previous Article | Next Article 
Molecular and Cellular Biology, September 2002, p. 6131-6141, Vol. 22, No. 17
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.17.6131-6141.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
Received 26 March 2002/ Returned for modification 7 May 2002/ Accepted 4 June 2002
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ABSTRACT |
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INTRODUCTION |
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131 (30). TFIIIC131 is the only subunit of TFIIIC that can be photo-cross-linked to DNA within the TFIIIB binding site (2), and its accessibility to photoprobes changes with the stepwise assembly of the preinitiation complex (18, 20). Protein-protein interactions between TFIIIC131 and the TFIIB-related subunit of TFIIIB, Brf1, initially enable only inefficient photo-cross-linking of TFIIIC131 to the upstream DNA. The efficiency of this photo-cross-linking increases significantly with the binding of TATA-binding protein (TBP) and formation of the B'-TFIIIC-DNA complex and then is diminished upon recruitment of the third TFIIIB subunit, Bdp1 (previously known as B" or TFIIIB90). These observations and the ability of TFIIIC to position TFIIIB at various distances upstream of the A block promoter element (16) are thought to reflect a series of conformational changes that occur in TFIIIC131 during preinitiation complex assembly (20). Conformational changes in the subunits of TFIIIB and the underlying DNA also occur during this process and confer high stability on the TFIIIB-DNA complex under a variety of solution conditions (4, 11, 14, 23, 27, 34). Additionally, deformation of the DNA by TFIIIB is thought to contribute to promoter opening by pol III (reference 22 and references therein). The activity of TFIIIC131 in pol III transcription is limiting both in vivo and in vitro and can be increased by dominant, gain-of-function mutations (42). Ten mutations of this type have been isolated by using a selection for suppressors of a promoter defect (A19) in the dimeric tRNA gene sup9-e A19-supS1 (33). These mutations map to a region of approximately 50 amino acids encompassing the second of 11 TPRs in TFIIIC131. The PCF1-1 mutation was the first of these dominant alleles to be isolated and has one of the strongest suppressor phenotypes in this group (33, 42). Early biochemical studies of the effect of the PCF1-1 mutation indicated that its ability to increase transcription was correlated with an increase in the activity of fractions containing TFIIIB (35, 41). This resulted from the preferential recovery of Brf1 in TFIIIB fractions from mutant cell extracts (33, 35). Subsequent studies of another TPR2 mutation, PCF1-2, employing entirely recombinant TFIIIB, showed that it increased transcription by facilitating the recruitment of Brf1 to TFIIIC-DNA (33). Under the solution conditions employed in this study, transcription and complex assembly with wild-type TFIIIC could not be driven to the same upper limit as was achieved with PCF1-2 TFIIIC. These data led to the proposal that the mutation facilitated a conformational change in TFIIIC131 that enabled Brf1 binding.
Since the initial genetic and biochemical characterization of PCF1-1 and PCF1-2, several high-resolution structures have been solved for functionally unrelated TPR proteins (1, 5, 9, 26, 37, 39). Each 34-amino-acid-long TPR comprises a pair of antiparallel 
helices (designated A and B) connected by a short turn. The similar packing of the helices within and between adjacent tandemly arranged TPRs generates a right-handed superhelix with a groove containing mostly side chains from the A helix. As demonstrated in several TPR-peptide cocrystal structures, the TPR groove provides or supports the ligand-binding surface (9, 26, 37). The PCF1-2 (T167I) and PCF1-1 (H190Y) mutations change residues in the A and B helices, respectively, that are not part of the consensus that defines the TPR fold (5). Thus, the different locations of these mutations suggest that they may activate pol III gene transcription by different mechanisms. In addition, studies showing that TFIIIIC131 plays a role in the recruitment of Bdp1 and interacts directly with this factor suggest an alternative step in TFIIIB complex assembly (other than Brf1 binding) that might be targeted by dominant PCF1 alleles (7, 36).
The interaction between TFIIIC131 and Brf1 has been demonstrated in two-hybrid experiments (3), in pull-down assays (24), and in solution interaction assays where it has been shown to proceed with the acquisition of helicity in one or both proteins (31). The amino-terminal half of TFIIIC131 contains two independent binding sites for Brf1: a high-affinity site in the amino terminus up to TPR5 (Nt-TPR5) and a lower-affinity site in TPR6 to TPR9. Interestingly, a larger fragment (Nt-TPR9) containing both of these sites has significantly lower affinity for Brf1 than either of the two smaller fragments. These findings demonstrate that autoinhibition of Brf1 binding sites in TFIIIC131 limits the interaction between these components in vitro. However, evidence that this phenomenon is biologically relevant for TFIIIC131 function has not yet been reported.
In this work, we have examined the mechanism of activation by PCF1-1. We show that the mutation (H190Y) increases the affinity of the interaction between TFIIIC-DNA and Brf1. However, site-directed mutagenesis experiments and binding studies with fragments of TFIIIC131 suggest that Brf1 does not make a direct contact with the side chain at amino acid 190 in TFIIIC131. Instead, dominant mutations at this position are shown to overcome the previously observed autoinhibition in the Brf1 binding reaction. The molecular basis of this effect is suggested to involve the stabilization of an intramolecular interaction in TFIIIC131 that promotes the interaction with Brf1.
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MATERIALS AND METHODS |
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3 Prime, Boulder, Colo.) was used per the manufacturer's directions. Degenerate oligonucleotides with the sequence CCTGGCAGCTXXXCTAAATGCATCCGATTGGG were used to mutagenize the plasmid pRS313PCF1+ at codon 190. Mutant plasmids were identified by the acquisition of an NsiI restriction site (underlined) introduced by silent mutagenesis. Candidate clones were retransformed into DH5, rescreened by Nsi1 digestion, and sequenced to confirm the identity of the amino acid substitution. PCF1 alleles on pRS313 were transformed into the yeast strain supAC1+ (see below), and the rescuing plasmid (pRS316PCF1+) was evicted on medium containing 5-fluoroorotic acid. Single colonies were obtained and assayed for viability and supS1 suppressor activity at 16, 30, and 37°C. Molecular modeling. Deep View software version 3.7b2 (12) was used to construct a model of TPR1 to TPR3 from TFIIIC131 based on the structure of protein phosphatase 5 (PP5 [5]). Deep View aligned the TPRs of the two proteins without any discontinuity and then threaded the sequence from TFIIIC131 onto the TPR structure of PP5 to generate the initial model. Subsequent energy minimization was performed with the GROMOS96 implementation in Deep View. Statistical analysis of structural models was performed with WHAT_CHECK software (http://www.cmbi.kun.nl: 1100/WIWWWI).
Proteins.
The purification of recombinant TFIIIB subunits (Brf1, TBP, and Bdp1) and yeast TFIIIC fractions has been described previously (4, 28, 31, 33). The specific activity of Brf1 in supporting single-round transcription was measured as described previously (28). For TFIIIC, whole-cell extracts from the S. cerevisiae strains supAC1+ (MAT ura3-52 pcf1
::LEU2 his3-11,15 leu2-3,112 trp1-1 met8-1 lys2::sup9eA19-supS1 pRS316 PCF1+) and supAC1-1 (same as supAC1+ but substituting pRS316 PCF1-1 [35]) were purified over four chromatographic columns (19, 23). Briefly, this involved step elution from BioRex70 and DEAE-Sephadex A25 and gradient elution from heparin-agarose and MonoQ columns. The resulting TFIIIC fractions had comparable DNA binding activities and generated apparent dissociation constants of 1.6 x 10-10 and 2.2 x 10-10 M for PCF1+ and PCF1-1, respectively. Western analysis for TFIIIC131 confirmed that the yield of TFIIIC was unaffected by the H190Y mutation (data not shown). Recombinant fragments of TFIIIC131 carrying the H190Y substitution were cloned, expressed, and purified as described elsewhere for their wild-type counterparts (32).
Two-hybrid assays. Interactions were assayed with Brf1 and Bdp1 in pASCHY2 and wild-type or mutant TFIIIC131 in pACTII (8). The reciprocal interactions between Brf1 in pACTII and TFIIIC131 in pASCHY2 were also measured. The resulting ß-galactosidase activity (units per milligram of protein) in the haploid yeast strain Y190 was measured after glass bead breakage (3, 8). Control experiments showed no ß-galactosidase activity, above that of the vector alone, in the absence of a partner protein.
Complex assembly assays. Complex assembly and electrophoresis on native polyacrylamide gels were performed with only minor modifications to previously described methods (31, 32). Unless otherwise indicated, reaction mixtures contained sup3-eST labeled DNA (10 fmol), yeast TFIIIC (5 fmol), Brf1 (6,000 fmol), TBP (250 fmol), and Bdp1 (100 fmol) with duplex poly(dG-dC) (25 µg/ml) as a nonspecific competitor DNA. For all experiments, TFIIIC-DNA complexes were preformed (20°C for 10 min) prior to addition of the other components. Reaction mixtures were then incubated at 20°C for an additional 60 min. In Fig. 3 to 5, duplex poly(dI-dC) (25 µg/ml) was used in place of duplex poly(dG-dC) to titrate trace amounts of TBP in the TFIIIC fractions or to limit the TBP concentration in the assembly of B'-TFIIIC-DNA complexes. The recruitment of Brf1 to mutant and wild-type TFIIIC-DNA complexes was examined in parallel experiments over a range of Brf1 concentrations from 25 to 900 nM. To assess the stability of Brf1-TFIIIC-DNA during electrophoresis, wild-type complexes were assembled and electrophoresed for 1, 2, 3, and 4 h. For Bdp1 titration experiments, template DNA (4 fmol), TFIIIC (0.3 to 2.5 fmol), Brf1 (6,000 fmol), and either 20 or 200 fmol of TBP were used to generate the substrate complex (B'-TFIIIC-DNA) for Bdp1 binding. The latter level of TBP drives higher-order complex formation essentially to completion with poly(dG-dC) as the nonspecific DNA. The recruitment of Bdp1 to mutant and wild-type complexes was examined in parallel over a range of concentrations from 0.5 to 20 nM. The ability of fragments of TFIIIC131 to bind to Brf1 in solution and thus inhibit TFIIIB-DNA complex formation was determined as previously described (31, 32). TFIIIC131 fragments were added to preformed TFIIIC-DNA complexes prior to the addition of the TFIIIB subunits. Mutant and wild-type fragments were always assayed in parallel. Individual pairwise comparisons (wild-type versus mutant) of inhibition isotherms generated with Nt-TPR9 and TPR1 to TPR9 always returned a higher apparent affinity (approximately twofold) for the mutant fragment. In contrast, individual experiments with mutant and wild-type Nt-TPR5 and TPR1-to-TPR5 fragments always generated inhibition isotherms that were identical within experimental error.
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To quantify the incorporation of Brf1 into TFIIIC-DNA complexes, the widths of the lines used for Peak Finder were set to exclude the edges of each lane (where peak trailing compromises band resolution). The curves generated by Peak Finder were then analyzed with the Peak Fitting software in Microcal Origin version 5.0, as follows. The curve parameters that best described the TFIIIC-DNA band were determined for the wild-type and mutant TFIIIC fraction in each experiment. In addition, the physical separation of TFIIIC-DNA and Brf1-TFIIIC-DNA complexes was determined from a lane in each titration where the band intensities of the two species were approximately equal. These parameters were used to define the shape and peak position of the two complexes for each lane in the corresponding Brf1 titration experiment. Two individual curves were then fitted for each curve generated by Peak Finder until the error was minimized. Brf1-TFIIIC-DNA complex formation, calculated as the fraction of the total number of TFIIIC-DNA complexes, was analyzed as described above. The quantitation of Brf1-TFIIIC-DNA complexes required that both TFIIIC-DNA and Brf1-TFIIIC-DNA bands be symmetrical (neither complex formed a trailing edge in the gel) and that no lane distortion occur during gel electrophoresis or drying.
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RESULTS |
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To quantify the assembly of TFIIIC-DNA and higher-order complexes, reactions at equilibrium were resolved on native polyacrylamide gels. Equal numbers of PCF1-1 and wild-type TFIIIC-DNA complexes were assembled based on the empirically determined DNA binding activities (Fig. 3, lanes 1 and 2). With the addition of a subsaturating amount of Brf1 (6,000 fmol), the mutant TFIIIC forms twofold more of the Brf1-TFIIIC-DNA complex than does wild-type TFIIIC (compare lanes 3 and 4). Consistent with this, it takes twice the amount of Brf1 (12,000 fmol) to generate similar levels of Brf1-TFIIIC-DNA complexes on wild-type TFIIIC as with mutant TFIIIC-DNA (lanes 4 and 5). Similar results are seen in the assembly of B'-TFIIIC-DNA (lanes 6 to 8), although the inability to resolve all of the complexes precludes their quantitation. Nonetheless, it is apparent that increasing the amount of Brf1 drives the assembly of higher-order complexes and diminishes the amount of wild-type TFIIIC-DNA (lanes 6 and 8). Differential binding of Brf1 to PCF1-1 and wild-type TFIIIC is also reflected in the number of TFIIIB-TFIIIC-DNA complexes (lanes 9 and 10) and heparin-resistant TFIIIB-DNA complexes (lanes 12 and 13). As will become apparent in later experiments, this in vitro differential in TFIIIB complex assembly is observed only under conditions where both Brf1 and TBP are limiting. That TBP is limiting under the conditions used in Fig. 3 is shown by the ability of additional TBP, but not Brf1 or Bdp1, to drive higher levels of TFIIIB-TFIIIC-DNA complex assembly with wild-type TFIIIC (compare lanes 11, 14, and 15 with lane 9). Additional Brf1 increases the level of Brf1-TFIIIC-DNA complexes (compare lanes 9 and 11), which accumulate rather than being converted into TFIIIB owing to the limited amount of TBP. Under these conditions, the addition of extra Bdp1 does not generate an increase in TFIIIB-TFIIIC-DNA complexes (compare lanes 9 and 15). It is, therefore, the addition of TBP in lane 14 that allows the assembly of higher levels of TFIIIB-TFIIIC-DNA complexes with wild-type TFIIIC (compare lane 9 to lane 14) and thus mimics the effect of the PCF1-1 mutation. Thus, as observed previously for the PCF1-2 mutation (33), PCF1-1 facilitates the recruitment of Brf1 to TFIIIC-DNA and allows the assembly of more TFIIIB complexes under conditions where both Brf1 and TBP are limiting.
Determination of an apparent binding affinity for the interaction of Brf1 with TFIIIC-DNA. Although the PCF1-1 mutation does not map to a TPR position that is known from structural studies to participate directly in ligand binding, the effect of the mutation on ligand (Brf1) binding affinity, be it direct or indirect, has not been assessed to date. Indeed, the affinity of Brf1 for wild-type TFIIIC-DNA has not yet been determined. In this regard, the relatively small amounts of TFIIIC that are available and the high nonspecific binding of Brf1 to solid supports (28) pose significant obstacles for a variety of quantitative methods. We, therefore, explored the feasibility of directly quantifying the interaction between TFIIIC-DNA and Brf1 by native gel electrophoresis. Owing to the limited separation of TFIIIC-DNA and Brf1-TFIIIC-DNA complexes, several modifications to our protocol for quantifying TFIIIB-DNA complexes (31, 32) were necessary in order to obtain binding curves from these experiments (see Materials and Methods). Most importantly, only gels of the highest quality (in band resolution and uniformity) were analyzed, and we incorporated the use of peak fitting software together with strict criteria for peak shape to quantify each species. As a first test of this approach, Brf1-TFIIIC-DNA complexes were assembled under identical conditions by using wild-type TFIIIC and electrophoresed in a native gel for 1, 2, 3, and 4 h (Fig. 4) (see Materials and Methods). Complexes electrophoresed for 1 h were not resolved sufficiently to allow quantitation of individual species. For the other time points, the formation of Brf1-TFIIIC-DNA was quantified as a fraction of the total number of TFIIIC-containing complexes. This analysis showed no significant variation in the fraction of Brf1-TFIIIC-DNA and yielded an average value of 49% ± 5%. Thus, the complexes appear to be relatively stable during electrophoresis.
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Further evidence that the PCF1-1 mutation facilitates the interaction between TFIIIC131 and Brf1 was obtained by using the yeast two-hybrid system. Interactions between wild-type TFIIIC131 and Brf1 are readily detected in this system by using ß-galactosidase activity as the reporter (3). However, the introduction of the PCF1-1 mutation into the Gal4 activation domain fusion generated a 26 ± 2-fold-higher level of ß-galactosidase activity than wild-type TFIIIC131 when assayed against Brf1 fused to the Gal4 DNA binding domain. The reciprocal fusion protein combination showed a 21 ± 5-fold enhancement of the mutant over wild type. Importantly, Gal4 DNA binding domain fusions of either TFIIIC131 (wild type or mutant) or Brf1 did not produce ß-galactosidase activity in the absence of the interacting partner (3; data not shown). These data confirm that the PCF1-1 mutation positively affects the interaction between TFIIIC131 and Brf1.
Analysis of Bdp1 binding to complexes containing PCF1-1 or wild-type TFIIIC.
Deletion of TPR2 (TPR2) from TFIIIC131 confers a temperature-sensitive phenotype in S. cerevisiae that is suppressed by overexpression of Bdp1 but not Brf1 or TBP (7). Similarly, the weak two-hybrid interaction between TFIIIC131 and Bdp1 is stimulated by TPR2 (36) while a negative effect of this deletion is seen on the interaction with Brf1 (3). As TFIIIC131 has recently been shown to interact directly with Bdp1 (7), the preceding findings suggest that dominant mutations like PCF1-1 in TPR2 may affect the recruitment of Bdp1 to the B'-TFIIIC-DNA complex. To address this possibility, we quantified the binding of Bdp1 to wild-type and PCF1-1 B'-TFIIIC-DNA complexes. Titrations of Bdp1 were performed on B'-TFIIIC-DNA complexes assembled with limiting amounts of Brf1 and TBP to maintain the differential recruitment of Brf1 (as in Fig. 3). The resulting TFIIIB-TFIIIC-DNA complexes, which are well resolved from the other species (e.g., Fig. 3), were quantified, and the data were plotted as a function of Bdp1 concentration. Binding isotherms obtained by nonlinear least squares analysis show that the mutant TFIIIC supports twofold-more TFIIIB complex formation than does wild-type TFIIIC (Fig. 7A ). From these initial fits, each titration was scaled to its upper endpoint and then refitted to the Hill equation. As shown in Fig. 7A (inset), the scaled wild-type and mutant isotherms are indistinguishable from one another. We then repeated these Bdp1 titrations in the presence of saturating levels of Brf1 and TBP. In contrast to the limiting conditions used previously, no differential in the level of TFIIIB complex formation was observed for wild-type and mutant TFIIIC under these conditions (Fig. 7B). The resulting binding isotherms demonstrate once again that the PCF1-1 mutation has no effect on the binding of Bdp1 (Fig. 7C); the wild-type B'-TFIIIC-DNA complex yielded an apparent affinity of 1.65 ± 0.14 nM with a Hill coefficient of 1.7 ± 0.2 whereas the complex containing PCF1-1 TFIIIC yielded 1.34 ± 0.15 nM with a Hill coefficient of 1.5 ± 0.2. As with the binding of Brf1 to TFIIIC-DNA, the high nonintegral value for the Hill coefficient is indicative of cooperative binding in the recruitment of Bdp1.
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The PCF1-1 mutation overcomes autoinhibition of Nt-TPR9 binding to Brf1. Previous biochemical studies have shown that a fragment of TFIIIC131 (Nt-TPR9) encompassing the hydrophilic amino terminus and the two TPR arrays (TPR1 to TPR5 and TPR6 to TPR9 together with the region between them) contains two Brf1 binding sites (32). Each TPR array binds independently to Brf1, and the inclusion of sequences amino terminal to TPR1 to TPR5 converts this otherwise weak binding fragment into one with high affinity for Brf1 (32). However, the relative Brf1 binding affinities of these fragments indicate that the Nt-TPR5 and TPR6-to-TPR9 sites are not fully accessible to Brf1 within the Nt-TPR9 fragment (32). Thus, the binding of Nt-TPR9 to Brf1 is autoinhibited. These findings suggest that the higher apparent affinity of Brf1 for PCF1-1 TFIIIC-DNA could be achieved either directly by an enhanced interaction with the TPR1-to-TPR5 binding site or indirectly through the relief of autoinhibition. To examine these possibilities, we assessed the affect of the PCF1-1 mutation on the Brf1 binding affinity of four TFIIIC131 fragments (Nt-TPR9, Nt-TPR5, TPR1 to TPR5, and TPR1 to TPR9) by a coupled equilibrium binding assay (31, 32). This assay monitors the inhibition of heparin-resistant TFIIIB-DNA complex formation that occurs when TFIIIC131 fragments compete with wild-type TFIIIC-DNA for binding to Brf1 in the presence of excess TBP and Bdp1. Each of the mutant TFIIIC131 fragments was purified to apparent homogeneity as described previously for their wild-type counterparts (32). Titrations of either mutant or wild-type TFIIIC131 fragments were then performed, the heparin-stripped TFIIIB-DNA complexes were resolved in native gels and quantified, and the data were analyzed to extract apparent dissociation constants for the various fragment-Brf1 interactions (32). The wild-type and mutant fragment binding isotherms from these experiments are compared in Fig. 8, and the apparent dissociation constants are listed in Table 1.
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In contrast to the preceding fragments, the mutant and wild-type TPR1-to-TPR9 fragments generate isotherms with Hill coefficients that deviate significantly from unity (Fig. 8 and Table 1). As outlined previously (32), these data suggest two possibilities: that the binding stoichiometry of the reaction has changed such that two molecules of TFIIIB70 are now bound for each molecule of TPR1 to TPR9 or that a single molecule of TFIIIB70 binds cooperatively to the two independent Brf1-binding domains in TPR1 to TPR9. In either case, the apparent affinities of the Brf1-TPR1-to-TPR9 complexes cannot be compared with the affinities of Brf1 complexes involving the other TFIIIC131 fragments. It is clear, however, that the PCF1-1 mutation increases the affinity of TPR1 to TPR9 for Brf1 (Table 1). For the same reasons as described above for Nt-TPR9, we conclude that the PCF1-1 mutation opposes the autoinhibition of Brf1 binding to TPR1 to TPR9.
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DISCUSSION |
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The preceding findings together with the results from site-directed mutagenesis at amino acid 190 suggest a structural model for activation by dominant, gain-of-function mutations at this position: these mutations, we propose, stabilize an alternative conformation of TFIIIC131 that promotes Brf1 binding. Given the properties of the wild-type and PCF1-1 fragments of TFIIIC131 (Table 1), the conformer favoring Brf1 binding is predicted to be stabilized by an intramolecular interaction between the tyrosine residue at amino acid 190 and some other site in the Nt-TPR9 fragment. Similar stabilizing interactions are likely for the other activating mutations at this position (Fig. 2). As noted above, a histidine residue at the position corresponding to amino acid 190 in TFIIIC131 is highly conserved in organisms from yeasts to humans and yet has intermediate phenotypic strength. Consistent with the above proposition, this residue presumably has a diminished ability to stabilize the alternative conformer. The biochemical reason that a restrained interaction is preferred is not clear. However, we speculate that the reversible or dynamic nature of the proposed alternative conformer is important for other functions of TFIIIC131. In support of this idea, we note that a specific internal deletion of BDP1 (bdp1-355-372), which by itself is temperature sensitive, is synthetically lethal when combined with PCF1-1 (15). Similarly, the more stringent requirements for both Brf1 and Bdp1 in TFIIIC-dependent complex assembly versus TBP-directed (TFIIIC-independent) assembly have been interpreted to reflect a need for interactions that reposition TFIIIC in order to allow TFIIIB complex assembly and other downstream steps in transcription to proceed (21, 25). Finally, mutations at amino acid 190 that fail to express the sup9-e A19-supS1 suppressor (and surely lack stabilizing intramolecular interactions proposed above) have no obvious growth defects on complete medium (Fig. 2). Cell growth under normal conditions, it seems, is not limited by the functional consequences of mutations at this site. In contrast, the ability to grow on selective medium requiring the expression of the supS1 suppressor is limited by the rate of transcription complex assembly stemming from the sup9-e A19 promoter defect. Mutations at amino acid 190 that increase the lifetime of the alternative, higher-affinity conformer are therefore able to increase the rate of complex assembly and transcription of the mutant promoter.
In agreement with earlier observations (33), we found that wild-type TFIIIC-DNA complexes could not be quantitatively converted into Brf1-containing complexes in the absence of TBP and Bdp1 (Fig. 3 and 5). This reaction appeared to reach saturation at about 80% conversion whereas it could be driven to completion with mutant TFIIIC (Fig. 6). A simple explanation for this result, consistent with the apparent Brf1 binding affinities (Fig. 6), is that wild-type Brf1-TFIIIC-DNA complexes may be less stable relative to complexes containing PCF1-1 TFIIIC and thus may dissociate during gel electrophoresis. However, our experiments were unable to detect in-gel dissociation of these complexes (Fig. 4). Nonetheless, it remains possible that about 20% of the wild-type Brf1-TFIIIC-DNA complexes may dissociate prior to entry into the gel. Alternatively, a conformationally distinct TFIIIC fraction may exist in which the Brf1 binding site is blocked (proposed initially in reference 33). Further evaluation of this behavior will require the development of new assays in this system.
Both halves of Brf1 interact with TFIIIC-DNA (21), and two-hybrid experiments indicate that the TFIIB-like half of Brf1 interacts with the amino terminus (Nt-TPR1) of TFIIIC131 (3). However, the regions of Brf1 that interact with the two independent binding sites in TFIIIC131 (Nt-TPR5 and TPR6 to TPR9) have not been defined biochemically. Moreover, it is not clear how Brf1 interacts with the large Nt-TPR9 fragment. In this regard, it is interesting that the apparent Brf1 binding constant for the mutant Nt-TPR9 fragment (192 ± 30 nM [Table 1]) is identical within experimental error to that for TPR6 to TPR9 (177 ± 27 nM [32]). Thus, the PCF1-1 mutation may relieve autoinhibition of Brf1 binding to TPR6 to TPR9. Of course, more complex interactions could also account for the data. In any event, it is certain that the high-affinity Brf1 binding site in Nt-TPR5 remains largely, if not entirely, inaccessible in the mutant Nt-TPR9 fragment. As noted previously for the wild-type fragment (32), this implies that Brf1 may bind sequentially to the two sites in TFIIIC131, within the context of TFIIIC. Given the apparent need for repositioning of TFIIIC131 in order to allow Bdp1 binding (21, 25), we suggest that the interactions between Brf1 and TFIIIC131 may be modified (e.g., extended or exchanged) at this stage in the assembly process.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant GM42728.
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FOOTNOTES |
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2% of the TFIIIB-TFIIIC-DNA complexes assembled in this assay.
