Both the N- and the C-terminal regions of Brf2 are required for U6 snRNA gene transcription.
The top of Fig. shows a comparison of the structures of human TFIIB, Brf1, and Brf2. These three proteins each contain a zinc-binding domain (shown in blue) and a core domain (shown in purple). Brf1 and Brf2 additionally contain unique C-terminal domains. To determine the role of different parts of Brf2, we synthesized the truncated Brf2 proteins illustrated in the lower part of Fig. . Brf2(66-419) lacks the region N-terminal to the core domain, which includes the Zn ribbon motif. The second truncation, Brf2(2-289), lacks the C-terminal domain of the protein. The full-length and truncated proteins all contain a histidine tag at their C terminus (indicated in red), which was used for purification after expression in E. coli
. The resulting proteins are shown in Fig. and, as revealed by immunoblot with an anti-Brf2 antibody after normalization of the amounts, in Fig.. These proteins were then tested for U6 transcription in a minimal in vitro transcription system, which consists of recombinant TBP, Bdp1, Brf2, and SNAPc
combined with highly purified Pol III from a HeLa cell line expressing tagged RPC4 (RPC53) (18
FIG. 1. Truncated Brf2 proteins lacking the regions N or C terminal to the core domain are inactive for U6 transcription. (A) Structure of human TFIIB, Brf1, and Brf2 and of the recombinant “full-length” (FL) and truncated Brf2 proteins expressed (more ...)
As seen in Fig. , TBP, Bdp1, and SNAPc combined with purified Pol III in the absence of Brf2 did not give rise to any detectable U6 transcript in the reconstituted in vitro transcription assay (lane 1). As expected, addition of recombinant full-length Brf2 resulted in efficient U6 transcription (lanes 2 to 4). However, when either Brf2(66-419) or Brf2(2-289) were used in place of full-length Brf2, U6 transcripts were not detectable (lanes 5 to 7 and lanes 8 to 10, respectively), suggesting that both of the regions N and C terminal to the Brf2 core domain are important for transcriptional activity.
Visualization by EMSA of the assembly of a U6 transcription preinitiation complex.
To enable us to study the functions of the N- and C-terminal regions of Brf2 in the formation of the transcription initiation complex, we set out to establish an electrophoretic mobility shift assay (EMSA) in which we could visualize the assembly of the various U6 transcription factors on the U6 promoter. We first focused on the Brf2-TFIIIB components. As shown in Fig. , unlike Brf2 or Bdp1, TBP on its own was able to form a protein-DNA complex on a probe containing the wild-type U6 PSE and TATA box (lanes 2 to 4). As observed previously, the addition of both TBP and Brf2 resulted in the formation of a prominent complex (henceforth referred to as the “two-factor complex,” labeled 2 in lane
5; see also Fig. , which summarizes the nomenclature used for some of the complexes), reflecting the cooperative binding of these two factors (6
). With TBP and Bdp1, a weak complex migrating much more slowly than the TBP complex was obtained (lane 6, complex labeled TBP/Bdp1). Since Bdp1 on its own does not bind detectably to the probe (lane 4), this suggests protein-protein interactions between Bdp1 and TBP on the DNA. With TBP, Brf2, and Bdp1, a robust complex migrating even more slowly than the TBP/Bdp1 complex was obtained (henceforth referred to as the “three-factor complex,” labeled 3 in lane 7; see Fig. ). This complex was supershifted by anti-Bdp1 and anti-TBP antibodies (which as expected did not bind to the probe on their own, see lanes 12 and 13), confirming that it contains Bdp1 and TBP (compare lanes 8 to 11 to lane 7). On the other hand, we did not observe a supershift with any of our anti-Brf2 antibodies (data not shown). Nevertheless, since the three-factor complex was not observed in the absence of Brf2 and migrated more slowly than the TBP/Bdp1 complex (compare lanes 6 and 7), it is highly likely that it indeed contains Brf2 but that the Brf2 epitopes recognized by the antibodies we tested are masked. None of the complexes were observed with a probe containing a mutated TATA box (lanes 15 to 17), indicating that they all depend on TBP-TATA box interactions.
FIG.2. Assembly of the U6 preinitiation complex. (A) Assembly of the Brf2-TFIIIB complex on the U6 TATA box. Either a probe with a wild-type mouse U6 PSE and human U6 TATA box (lanes 1 to 13) or a wild-type mouse PSE and a mutant TATA box (lanes 14 to 17) was (more ...)
We next combined the Brf2-TFIIIB components with SNAPc. As shown in Fig. , as we titrated increasing amounts of SNAPc into a binding reaction containing the three Brf2-TFIIIB components, we observed a gradual increase in the formation of a large complex (henceforth referred to as the “four-factor complex,” labeled 4 in lane 4; see Fig. ) migrating more slowly than the three-factor complex containing TBP, Brf2, and Bdp1 (compare complex 4 in lanes 4 to 6 with complex 3 in lane 1). This complex was not observed in the absence of the Brf2-TFIIIB components (lanes 7 to 11) and could be supershifted with anti-TBP, anti-Bdp1, and anti-SNAP190 antibodies (Fig. , compare lanes 3 to 8 with lane 2), confirming that it contains TBP, Bdp1, and SNAPc. Like complex 3, complex 4 was not supershifted by the anti-Brf2 antibodies we tested (data not shown), most likely because within the complex, the relevant Brf2 epitopes are not accessible to the antibodies.
In addition to complex 4, binding reactions containing SNAPc
and Brf2-TFIIIB gave rise to four prominent smaller complexes (see, for example, Fig. , lane 5). As shown in Fig., comparison with binding reactions containing various subsets of the four components on either a wild-type probe or a probe with a mutated TATA box indicated that, of these four smaller complexes, the one migrating the fastest is the two-factor complex containing TBP and Brf2 (compare lanes 3 and 6, complex 2, see also lanes 8 to 10), whereas the one above it corresponds to just SNAPc
bound to the DNA (compare lanes 1 and 6; see also lanes 4, 7, 9, and 10). The third complex from the top in lane 6 comigrates with one formed by TBP, Brf2, and SNAPc
(compare lanes 4 and 6, complex labeled with a white circle; see also lanes 9 and 10), whereas the complex just below the four-factor complex in lane 6 corresponds to the three-factor complex (compare lanes 5 and 6, see also lanes 8 to 10). Thus, by EMSA we can visualize a complex containing the basal transcription factors SNAPc
and Brf2-TFIIIB, which are known to be sufficient to reconstitute U6 transcription when combined with a highly purified Pol III complex (18
), as well as visualize various subcomplexes.
The C-terminal region of Brf2, but not the N-terminal region, is required for binding with TBP and interacting with SNAPc on the U6 snRNA gene promoter.
We used the EMSA system described above to determine whether the N- and C-terminal regions of Brf2 might be involved in initiation complex assembly. In the interest of space, all EMSA experiments presented hereafter show only the region corresponding to the upper half of the gel containing the various complexes. Lanes 1, 2, and 3 in Fig. show the two-, three-, and four-factor complexes (containing, respectively, TBP and Brf2; TBP, Brf2 and Bdp1; and TBP, Brf2, Bdp1 and SNAPc
) obtained with full-length Brf2 (complexes labeled 2, 3, and 4). With the Brf2(66-419) truncated protein, the two-, three-, and four-factor complexes were all observed (lanes 10 to 12), indicating that the region N-terminal to the core domain is not required for binding to the TBP-TATA box complex (lane 10), recruitment of Bdp1 (lane 11), or assembly of the full complex (lane 12). In contrast, with a Brf2(66-289) truncated protein containing just the Brf2 core domain and with the Brf2(2-289) truncated protein, none of these complexes were obtained (lanes 4 to 9). In particular, we did not observe a two-component complex containing TBP and a truncated Brf2 (lanes 4 and 7). The only complexes observed were those containing either just SNAPc
(compare lanes 6 and 9 with lane 16) or SNAPc
and TBP (compare lanes 6 and 9 with lane 13), a finding consistent with previous observations that SNAPc
and TBP bind cooperatively to the U6 promoter (27
). Thus, the Brf2 region C-terminal of the core domain is required for detectable binding of Brf2 together with TBP to the U6 TATA box in the EMSA. Moreover, it is required for the formation of all subsequent complexes containing both Brf2 and TBP.
FIG. 3. Activity of truncated versions of Brf2 in preinitiation complex assembly. (A) The C-terminal region of Brf2 is required for binding to the TBP/TATA box complex, whereas the N-terminal region is not required for preinitiation complex assembly. The probe (more ...)
Brf2, which does not bind to DNA on its own, can be recruited to the U6 promoter not only through protein-protein interactions with TBP binding to the TATA box but also through protein-protein interactions with SNAPc
binding to the PSE (16
). However, with the truncated Brf2 proteins missing the C-terminal region, we did not observe novel complexes that might contain Brf2 but lack TBP. We wondered, therefore, whether the C-terminal region of Brf2 might be required for cooperative binding with SNAPc
. As shown in Fig. , when only SNAPc
and Brf2 were added to the U6 promoter probe, we observed a weak complex migrating more slowly than the SNAPc
-only complex and slightly faster than the TBP/SNAPc
complex (lane 4, complex labeled with a white arrowhead, compare with lanes 1 and 3). This complex most likely contains SNAPc
and Brf2 and presumably forms due to interactions between Brf2 and the PSE-bound SNAPc
. Notably, a similar, even darker complex was observed when full-length Brf2 was replaced by Brf2(66-419) but not when it was replaced by Brf2(2-289) (compare lane 4 with lanes 5 and 6). Thus, the C-terminal region of Brf2 is not only required for cooperative binding with TBP bound to the TATA box but also for cooperative binding with SNAPc
bound to the PSE.
These results indicate that the C-terminal domain of Brf2 is necessary for the protein to form a complex with TBP, as well as with SNAPc, on a U6 promoter. Because of the failure to form these initial complexes, higher-order complexes in turn cannot form. Thus, the inability of Brf2(2-289) to direct U6 transcription (Fig. above) can be ascribed to a failure of transcription initiation complex assembly. On the other hand, the inability of Brf2(66-419) to direct U6 transcription probably results from a defect at a step after transcription factor assembly, such as Pol III recruitment or promoter opening.
As discussed above, Brf2 and TFIIB have several features in common but they are used by different RNA polymerases—TFIIB by Pol II and Brf2 by Pol III. To learn more about the functions of the various Brf2 regions and the determinants of RNA polymerase specificity, we constructed a set of TFIIB/Brf2 chimeric proteins, which are shown in Fig. . We divided Brf2 into core region and regions N and C terminal to the core and TFIIB into core region and region N terminal to the core and constructed four chimeras by swapping these regions between the two proteins. The chimeras were named based on the origin of the regions from which they were comprised. Thus, for example, 2/B/− consists of the N-terminal region of Brf2, the core domain of TFIIB, and no C-terminal domain, whereas B/2/2 consists of the N-terminal region of TFIIB and the core and C-terminal domains of Brf2. The four chimeras, as well as full-length Brf2 and TFIIB, were expressed in E. coli and partially purified through a C-terminal His tag. The resulting proteins are shown in Fig. as visualized by staining with the GelCode Blue Stain reagent (Pierce) and in Fig. as visualized by immunoblot with an anti-His tag antibody after normalization of the amounts.
FIG.4. Brf2-TFIIB chimeric proteins. (A) The structures of the various chimeras are shown. The numbers under the boxes correspond to amino acids. The C-terminal His tags are indicated in red, and the N-terminal FLAG tags are indicated in blue. (B) The chimeras (more ...)
We then tested the four chimeras for U6 transcription in the minimal in vitro transcription system, and the results are shown in Fig. . Combining full-length Brf2, but not full-length TFIIB, with TBP, Bdp1, SNAPc
, and purified Pol III reconstituted U6 transcription (compare lanes 2 to 4 to lanes 5 to 7 and to lane 1), consistent with previous observations showing that TFIIB is not active for Pol III-directed U6 transcription (26
). Both the 2/B/− and B/2/− chimeras were inactive in this assay (data not shown) as was the B/2/2 chimera (lanes 8 to 10). The 2/B/2 chimera showed barely detectable activity (lanes 11 to 13). This prompted us to widen our titration of the 2/B/2 chimera, but in all cases the U6 signal was extremely weak (lanes 15 to 19), although clearly detectable in the original film. Thus, the chimeras are generally inactive for U6 transcription, except for the 2/B/2 protein, which displays extremely weak activity.
TFIIB cannot recruit Bdp1 to the U6 promoter.
To understand the defects of TFIIB in terms of U6 transcription activity, as well as those of the chimeric proteins, we tested their capacity to assemble a U6 preinitiation complex by using EMSA. Figure , lanes 1 to 6, show the locations of the SNAPc complex alone (lane 1), the two-, three-, and four-factor complexes (lanes 3, 5, and 6) and the complexes containing TBP and SNAPc (lane 4, complex labeled TBP/SNAPc) and TBP, Brf2, and SNAPc (lane 4, complex labeled with a white dot on the left). We first focused on TFIIB. As expected, TFIIB alone was unable to bind to the probe but bound cooperatively with TBP to the U6 TATA box (lanes 7 and 8). However, upon addition of Bdp1, no additional complex was observed (lane 9), indicating that the TBP/TFIIB complex cannot recruit Bdp1. Interestingly, when TBP, TFIIB, Bdp1, and SNAPc were present in the binding reaction, we obtained a complex containing TBP, TFIIB, and SNAPc (lane 10, complex labeled with a white dot on the left). Thus, on the Pol III U6 promoter, TFIIB can assemble efficiently with TBP and SNAPc but is unable to recruit Bdp1.
FIG. 5. Activity of Brf2-TFIIB chimeras in U6 preinitiation complex assembly. (A) Activity of TFIIB and chimeras containing the C-terminal region of Brf2. The probe contained a wild-type human PSE and a human TATA box. Where indicated above the lanes, the binding (more ...) The B/2/− chimera does not bind with TBP to the U6 promoter, whereas the 2/B/− chimera does not recruit Bdp1.
We next examined the B/2/− and 2/B/− chimeras. In Fig. , the locations of the two-, three- and four-factor complexes in the EMSA are shown (lanes 1 to 3), as are those of the complexes containing just SNAPc
(lane 13); TBP and SNAPc
(lane 10, complex labeled TBP/SNAPc
on the left of the panel); and TBP, Brf2, and SNAPc
(lane 11, complex labeled with a white dot on the left). The B/2/− chimeric protein contains the core of Brf2 but lacks its C-terminal region. Consistent with the deletion results obtained above, which indicate that the Brf2 C-terminal region is necessary for efficient cooperative binding with TBP, as well as with SNAPc
(Fig. ), the B/2/− chimera was unable to form any of the Brf2-dependent complexes (lanes 4 to 6). The only complexes visible were those containing either just SNAPc
or both SNAPc
and TBP (see lane 6). With the 2/B/− chimeric protein, which contains the core domain of TFIIB, we observed a two-factor complex containing TBP and 2/B/− (lane 7), a finding consistent with the known ability of the TFIIB core to bind to a TBP-TATA box complex (3
). This complex was, however, unable to recruit Bdp1 (lane 8), although it could assemble with SNAPc
to form a complex containing TBP, 2/B/−, and SNAPc (lane 9, complex labeled with a white dot). This is consistent with the inability of full-length TFIIB to recruit Bdp1 (Fig. ). Moreover, the results obtained above with Brf2(66-419) (Fig. ) indicate that the N-terminal region of Brf2 is not required for Bdp1 recruitment but do not address the possibility that this region is capable of recruiting Bdp1 in a manner redundant with the core or C-terminal regions of Brf2. The results with the 2/B/− chimera show that the N terminus of Brf2 cannot recruit Bdp1 when targeted to a TBP-TATA box complex.
Chimeric Brf2 proteins containing the N-terminal or core region of TFIIB can support assembly of the four-factor complex.
The activities of the B/2/− and 2/B/− chimeras in transcription complex assembly indicated an essential role for the Brf2 C-terminal domain in binding to the TBP-TATA box complex and perhaps in recruiting Bdp1. We therefore tested in the EMSA the B/2/2 and 2/B/2 chimeras, which differ from B/2/− and 2/B/− only by addition of the Brf2 C-terminal domain. As shown in Fig. , both proteins were capable of forming a complex with TBP on the U6 TATA box (lanes 12 and 16, complex 2), although the complex formed with B/2/2 was weaker than that obtained with either Brf2 (lane 3) or 2/B/2. This is surprising since Brf2(66-419), which lacks the N-terminal region of Brf2 altogether, binds as efficiently as full-length Brf2 to a TBP/TATA box complex (Fig. ). Moreover, as shown below, B/2/2 binds as efficiently as Brf2 to TBP in a GST pull-down assay. These differences may indicate that the presence of the N-terminal region of TFIIB in place of that of Brf2 somehow reduces the B/2/2 chimera's ability to assemble with TBP on the U6 TATA box but not off the DNA. We have not, however, pursued this observation further. For the 2/B/2 chimera, its binding to the TBP-TATA box complex may be due to the TFIIB core region, the Brf2 C-terminal region, or both since these two regions both contain TBP-binding activity. However, as with B/2/2, the two-factor complex obtained with 2/B/2 migrated as a tight band close to the position observed with full-length Brf2 (compare lanes 12 and 16 with lane 3) and very different from the diffuse and slower-migrating complex observed with TFIIB and TBP (lane 8). Although we do not know what causes the TFIIB-TBP-TATA box complex to be diffuse, this suggests that 2/B/2 binds to the TBP-TATA box complex in a manner more similar to Brf2 than TFIIB.
The promoter-bound B/2/2 and 2/B/2 proteins could recruit Bdp1 (Fig. , lanes 13 and 17, complex 3) and SNAPc (lanes 14 and 18, complex 4), although the recruitment of SNAPc by 2/B/2 is not efficient (lane 18). The B/2/2 protein's ability to promote assembly of the four-factor complex is consistent with the results above (Fig. ) showing that the core and C-terminal regions of Brf2 together are sufficient for interaction with TBP, Bdp1, and SNAPc on the DNA. Its inactivity in U6 transcription indicates that the essential function(s) of the N-terminal region of Brf2 cannot be efficiently performed by the N-terminal region of TFIIB. In the case of the 2/B/2 protein, its ability to recruit Bdp1 contrasts with the inability of 2/B/− to perform this same function and indicates that the Brf2 C-terminal region is required for Bdp1 binding. Moreover, the formation of a four-factor complex with 2/B/2 indicates that when the C-terminal region of Brf2 is present the core region of Brf2 can be replaced with that of TFIIB. Indeed, the 2/B/2 chimera is capable of directing a very low level of U6 transcription (Fig.). However, the efficiency of both four-factor complex assembly and transcription is very low, indicating that either the TFIIB core region is not placed optimally for full function in the chimeric protein or that there is some function of the Brf2 core region that cannot be fully replaced by the TFIIB core region. One possibility is that the interactions between the 2/B/2 chimera and both Bdp1 and SNAPc together may not be stable enough to support efficient reconstituted U6 in vitro transcription. Another possibility is that the core domain of Brf2 is involved in Pol III recruitment, which was not assayed in our EMSA experiments.
The C-terminal region of Brf2 is required for binding to TBP off DNA.
To test which Brf2 region is required to bind to TBP off the promoter DNA, we performed the GST pull-down experiment shown in Fig. . GST-TBP, as well as the negative controls GST and GST-protein carboxyl methyltransferase (GST-PCMT), were immobilized on glutathione-agarose beads, and the binding of various histidine-tagged proteins to these beads was visualized by immunoblotting with anti-HT antibodies. Lanes 1 to 4 show the proteins tested, namely, TFIIB, Brf2, B/2/−, and B/2/2. Both TFIIB and Brf2 bound specifically to GST-TBP in this assay, with Brf2 binding even more strongly than TFIIB (lanes 5 to 8). Strikingly, however, the B/2/− chimera, which contains the Brf2 core but not the Brf2 C-terminal region, bound very weakly to GST-TBP (lane 11), although the signal was clearly above the background seen with both GST alone and GST-PCMT (lanes 9 and 10). With the B/2/2 chimera, which contains the Brf2 C-terminal domain, very efficient and specific binding to GST-TBP was observed (lanes 12 to 14). These results provide further evidence for the crucial role of the C-terminal region of Brf2 in TBP binding and show that the region performs this function even off the promoter DNA.
FIG. 6. Binding of Brf2, TFIIB, and chimeric proteins to TBP off DNA. GST alone, GST-TBP, or GST-PCMT were immobilized on glutathione-agarose beads as indicated above lanes 5 to 14. 3 μg of TFIIB, Brf2, B/2/−, or B/2/2 were then mixed with 10 (more ...)