©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Complex Interactions between Yeast TFIIIB and TFIIIC (*)

Nathalie Chaussivert , Christine Conesa , Salam Shaaban (§) , André Sentenac (¶)

From the (1)Service de Biochimie et Génétique Moléculaire, CEA-Saclay, F91191 Gif sur Yvette Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transcription of yeast class III genes requires the sequential assembly of the general transcription factors TFIIIC and TFIIIB, and of RNA polymerase III, into an initiation complex composed of at least 25 polypeptides. The 70-kDa subunit of TFIIIB (TFIIIB70) is central in this network of interactions as it contacts both TATA-binding protein and a subunit of polymerase III. We show here that the TATA-binding protein interacts with the carboxyl-terminal part of TFIIIB70. TFIIIB70 also contacts TFIIIC (factor ) via its 131 subunit. The protein domains of 131 and TFIIIB70 involved in this interaction, either positively or negatively, were mapped using the two-hybrid system. We provide evidence that intramolecular interactions mask functional domains in both polypeptides.


INTRODUCTION

The cascade of interactions leading to class III gene activation and the key components involved in this process have been much investigated(1, 2, 3, 4) . The yeast factor TFIIIC or (six subunits, named 138, 131, 95, 91, 60, and 50), plays a primary role in the process of transcription complex assembly by binding to the intragenic promotor sequences of tRNA genes or to the preformed 5 S DNATFIIIA complex. Once bound, TFIIIC acts as an assembly factor to allow the binding of the initiation factor TFIIIB (three components, TATA-binding protein (TBP),()TFIIIB70 and TFIIIB90) to an upstream gene position. TFIIIB then recruits RNA polymerase III (pol III) and directs accurate initiation of transcription(5, 6) . The genes encoding most of these components are cloned(4) .

We have shown previously that TFIIIB70 interacts with TBP (7) and with the C34 subunit of pol III(8) . It also interacts with TFIIICDNA complex, a weak interaction profoundly modified by addition of TBP(6) . Thus, TFIIIB70 appears as a central bridging factor between the basal components of the class III transcription machinery, in a way that is much similar to the role proposed for TFIIB in the case of class II genes(9) . TFIIB is a target of transactivators (10, 11, 12) that facilitate its incorporation into the early preinitiation complex; there it binds TBP (and possibly DNA) and recruits the RNA polymerase IITFIIF complex(13, 14, 15, 16) . Indeed, TFIIB and TFIIIB70 have a conserved core with a NH-terminal region which contains a putative zinc finger domain, followed by two imperfect direct repeats (17-22) that, in TFIIB, harbor the target for TBP(13, 14, 15, 16) . A major structural difference is the existence in TFIIIB70 of a carboxyl-terminal extension (CTE) that almost doubles the size of the polypeptide compared to TFIIB (see Fig. 4; Refs. 20-22). One hypothesis is that the conserved domains were retained to perform basal functions like TBP, DNA, and polymerase binding, whereas the CTE evolved to recognize the assembly factor TFIIIC. The interaction with TFIIIC is of particular interest as it confers gene specificity. Using the two-hybrid system, we have sought first to identify the subunit(s) of TFIIIC that interacts with TFIIIB70, then to map the protein domains involved in this interaction. Unexpectedly, it was the TFIIB-like region that we found to interact with TFIIIC, via its 131 subunit. We also show that the CTE of TFIIIB70 is involved in TBP binding.


Figure 4: Schematic representation of mutant TFIIIB70 fusion proteins. Parts of the open reading frame encoding TFIIIB70 were amplified by polymerase chain reaction and fused in frame with the GAL4 DNA binding or activation domains. Numbers in parentheses indicate the TFIIIB70 amino acids present in the fusion protein. Motifs of TFIIIB70 homologous to TFIIB are indicated.




MATERIALS AND METHODS

Construction of Yeast Strains

Standard genetic techniques and media were used. Plasmids harboring modified alleles of the TFC4 gene were used to transform the YCK107 haploid strain containing the TFC4 gene on a plasmid with the chromosomal copy disrupted(23) . The modified copies of TFC4 were substituted for wild type TFC4 by plasmid shuffling on plates containing 5-fluoroorotic acid. Viable strains isolated at 30 °C have also been tested for growth at 37 and 16 °C.

Site-directed Mutagenesis and Construction of Plasmids

Oligonucleotide-mediated mutagenesis was performed as described by the manufacturer, using a Muta-Gene kit (Bio-Rad). Uracil-enriched single-stranded DNA was prepared from a Escherichia coli strain CJ236.

Single-stranded pCK14 (23) or pNC1 DNA was used to mutagenize the TFC4 gene. pNC1 plasmid was constructed by introducing a BamHI site at -9 relative to the TFC4 ATG codon and by destroying the BamHI site at +2310. pNC8 was constructed by modifying the BamHI site of pNC1 from -9 to -8 and destroying the EcoRV site at +2710. pNC11 to pNC31 were obtained by deletion of nucleotides 4-369, 4-292, 382-483, 484-585, 586-687, 688-789, 790-801, 1294-1398, 1396-1503, 1500-1605, 1606-1707, 1810-1881, 1830-1881, 1882-1920, 1921-2034, 1921-1977, 1977-2034, 2035-2094, 2077-2163, 2623-2724, and 2975-2986 of TFC4, respectively. Single-stranded pNC1 DNA was used to construct pNC13, pNC14, pNC22, pNC26, pNC27, and pNC29. The KpnI/SpeI fragments from plasmids pNC11 to pNC31 were cloned into pUN45.

Single-stranded pRMS3 DNA (22) was used to mutagenize the PCF4 gene. pRMS3-50 was created by introducing a NcoI site at -8 relative to the PCF4 ATG codon. pRMS3-50,52 and pRMS3-52,54 were created by introducing a NcoI site at -8 and +107, respectively, and a stop codon at +859. pRMS3-53 and pRMS3-54 were created by introducing a NcoI site at +752 and +107, respectively. The EcoRI, BglII/EcoRI, and NdeI/SalI fragments of pRMS3 were cloned in pET28b (Novagen), leading to plasmids pCC-7, pCC-8 and pCC-9, respectively.

Construction of GAL4 Fusions

pAS-CC and pACT-CC were obtained by digestion of pAS2 (24) or pACTII (kindly provided by S. Elledge) by NcoI, fill-in by Klenow DNA polymerase, and religation. pACT-JR, a gift from J. Rüth, was constructed by digestion of pACT-CC by XmaI, fill-in by Klenow enzyme, and religation. The 3382-base pair BamHI fragment from pNC8 harboring the TFC4 open reading frame was cloned into pAS2 for fusion with GAL4-(1-147) or into pACTII for fusion with GAL4-(768-881). pAS-131 was then digested by NdeI and religated to construct the GAL4-(1-147)-N3 fusion. The GAL4-(768-881)-N3 fusion was obtained by digestion of pNC1 by NdeI, fill-in by Klenow enzyme, digestion by BamHI, and cloning of the resulting fragment into pACTII. The 1065-base pair BamHI fragment from pCK14 was cloned into pAS-CC and pACT-CC to obtain GAL4-N4 fusions. The BamHI fragments of pNC13, pNC14, and pNC22 were cloned into pACT-JR to obtain the GAL4-TPR1, GAL4-TPR2, and GAL4-basic2 fusions, respectively. The TFC4 open reading frame, deleted of nucleotides 4-292 or 586-687, was cloned into the BamHI site of pACTII to obtain the GAL4-(768-881)-N2 and TPR3 fusions, respectively.

The GAL4-131-0TPR, GAL4-131-1TPR, GAL4-131-5TPR, GAL4-131-9TPR, and GAL4-bHLH fusions were constructed by polymerase chain reaction amplification of nucleotides 1-300, 1-495, 1-912, 1-1737, and 1727-2205 of pCK14, respectively. The oligonucleotides primers were designed in order to create a NcoI site at -8 and a BamHI site after the stop codon at +303, +498, +915, +1740, and +489, respectively. The resulting NcoI/BamHI fragments were then cloned into pAS2 and pACTII. The GAL4-TFIIIB70 fusions were constructed by cloning the NcoI/SalI fragments from pRMS3-50, pRMS3-50,52, pRMS3-53, pRMS3-54, and pRMS3-52,54 into pAS2 and pACTII. The sequence of the fusion joint was determined for all the constructs. The plasmids have been used to transform a Y526 yeast strain.()

GAL1-lacZ Activation Assay

Seven independent transformants for each combination of plasmids were grown as patches for 2 days at 30 °C on synthetic complete solid medium containing 2% raffinose as carbon source. -Galactosidase activity was revealed by overlaying the cells with 10 ml of X-gal agar as described (8) and incubating the plates for 24 h at 30 °C. -Galactosidase activity was measured in yeast extracts exactly as described previously(8) .

Expression of TFIIIB70 Variants

Recombinant histidine-tagged TFIIIB70 variants were expressed in E. coli cells from plasmids pSH360 (a gift from Steve Hahn), pCC-7, pCC-8, and pCC-9, purified by chromatography on Ni-nitrilo-triacetic acid-agarose (Qiagen) under native conditions.()pCC-7, pCC-8, and pCC-9 encoded TFIIIB70 derivatives from residues 263 to 596 (CTE), 14 to 262 (CTE), or 141 to 596 (N140), respectively, tagged at their NH-terminal end with six histidine residues and the T7-TAG epitope (Novagen).

Interaction of TFIIIB70 Variants with S-Labeled TBP

FarWestern analysis was performed as described using in vitro synthesized S-labeled TBP(7) . Recombinant TFIIIB70 derivatives were separated by SDS-PAGE and transferred on a nitrocellulose membrane. The filters were incubated with S-labeled TBP, washed, and autoradiographed. TFIIIB70 polypeptides were located by anti-TFIIIB70 antibodies (kindly provided by Steve Hahn) or by anti-T7-TAG antibodies (Novagen). Immune complexes were visualized with antibodies tagged with alkaline phosphatase (Promega).


RESULTS

TBP Interacts with the CTE Moiety of TFIIIB70

Using a FarWestern blotting experiment, we have previously demonstrated that TBP interacted with TFIIIB70 in absence of DNA(7) . To delineate more precisely the protein domains involved in this interaction, three derivatives of TFIIIB70 were expressed in E. coli cells, as histidine fusions, and purified under native conditions on Ni-nitrilo-triacetic acid-agarose. After SDS-PAGE, the proteins were transferred onto a membrane and probed with S-labeled TBP. As shown in Fig. 1(lanes 3 and 4), the CTE moiety of TFIIIB70, migrating as a polypeptide of 49 kDa, bound TPB. A COOH-terminal truncated form of the CTE migrating as a 41-kDa polypeptide (this polypeptide was recognized by antibodies directed to the T7-TAG epitope, located at the NH-terminal end of the recombinant CTE construct, not shown) bound very weakly to TBP, as judged from the relative band intensity in the Western blot and in the autoradiogram, suggesting that the integrity of the CTE was important for TBP binding. In contrast, the integrity of the TFIIB-like moiety did not seem necessary, since a derivative of TFIIIB70 deleted at the NH-terminal end (N140, lacking the zinc finger motif and half of the first repeated domain) was able to bind TBP (lanes 5 and 6). In fact, no binding of TBP could be detected with the TFIIB-like domain of TFIIIB70 (CTE, lane 8), migrating as a polypeptide of 33 kDa (lane 7). These results indicated that the CTE moiety of TFIIIB70 was involved in TBP binding. Note that a COOH-terminal truncated form of TBP, lacking the last 14 amino acid residues, was not able, when labeled with [S]methionine, to bind to TFIIIB70 polypeptides (data not shown).


Figure 1: TBP interacts with the CTE domain of TFIIIB70. Recombinant TFIIIB70 (WT) or TFIIIB70 derivatives comprising residues 263-596 (CTE), 141-596 (N140), or 14-262 (CTE), expressed as hexahistidine fusions, were purified from E. coli cells under native conditions. Eluted polypeptides (1-2 µg) were subjected to SDS-PAGE, transferred onto a membrane, and probed with S-labeled TBP as described under ``Materials and Methods.'' Labeled polypeptides were revealed by autoradiography (lanes 2, 4, 6, 8). The same membrane was then incubated with an anti-TFIIIB70 antiserum (lanes 1, 3, 5) or with antibodies directed to the T7-TAG epitope (lane 7), and immune complexes were visualized using antibodies tagged with alkaline phosphatase. The molecular weight of the major polypeptides contained in wild type (left) or deleted (right) TFIIIB70 fractions is indicated.



Genetic Deletion Analysis of 131

Topological studies of TFIIICDNA and TFIIIBTFIIICDNA complexes showed that only 131 subunit was located upstream of the transcription start site. This upstream location therefore made 131 a likely candidate for assembling TFIIIB through interactions with TFIIIB70(25, 26, 27) . 131 polypeptide, encoded by TFC4, comprises a series of notable motifs, including a highly charged NH-terminal domain, a motif akin to basic-helix-loop-helix-zipper (bHLH-Zip), and 11 tetratricopeptide repeat (TPR) units divided in three blocks(23) . Using genetic deletion analysis, we first investigated in vivo the importance of these domains (Fig. 2). The motifs were deleted individually, and centromeric plasmids harboring the mutant copies of the TFC4 gene were tested for their ability to functionally replace, at different temperatures, a chromosomally disrupted copy of TFC4. As shown in Fig. 2, deletion of the NH-terminal domain (N1, N2), of all but two TPR units, and of the loop of the bHLH-Zip motif (loop), led to a lethal phenotype. Three deletions resulted in a temperature-sensitive phenotype (TPR8, basic2, H1). Deletion of the TPR9, of several domains of the bHLH-Zip motif (basic1, H1, zipper), had no effect on cell growth. This deletion analysis suggested that most of the characteristic motifs were important for the structural or functional integrity of 131.


Figure 2: Deletion analysis of 131. The motifs noted in 131 protein were deleted individually using polymerase chain reaction amplification or directed mutagenesis of the TFC4 gene. The positions of deleted amino acids (inclusive) are indicated for each construct. Centromeric plasmids harboring a deleted copy of TFC4, expressed from its own promoter, were tested for their ability to functionally replace, at different temperatures, a chromosomally disrupted copy of TFC4. Lethal (-), wild type (+), and temper-ature-sensitive (ts) phenotypes are indicated.



Extragenic Suppression of 131 Mutants

We used the three temperature-sensitive mutant strains (TPR8, basic2, H1) to uncover potential genetic interactions of TFIIIC with components of the class III transcription apparatus. Overexpression of TFIIIB70, TBP, and 95 harbored on multicopy plasmids was found to suppress the temperature-sensitive phenotype of the three 131 mutant strains. These results confirmed the genetic interaction of 131 with TFIIIB70 (28) and suggested a network of interactions between 131, two components of TFIIIB and 95 (95 subunit is thought to interact with block A(1, 2, 3, 4) ). However, increased gene dosage of TFIIIB70, TBP, and 95 was also reported to suppress a mutation in 138, the B-block binding subunit of TFIIIC(29) . Therefore, these suppression effects do not demonstrate direct interactions between TFIIIB and TFIIIC components. Hence, increased concentration of TPB or TFIIIB70 could influence the rate of TFIIIB assembly or the rate of transcription complex formation on class III genes.

131 Interacts with TFIIIB70

To demonstrate a direct interaction of 131 with TFIIIB components, we used the two-hybrid system that detects in vivo interactions between two proteins overproduced in yeast cells(30, 31) . The proteins are fused to the DNA binding domain, or to the transcriptional activation domain, of the yeast GAL4 protein. If the two proteins interact, a chimeric GAL4 protein is reconstituted that activates the transcription of a lacZ reporter gene. This method previously revealed interactions between TFIIIB70 and the pol III subunit C34(8) . The open reading frames encoding TFIIIB70, TBP, TFIIIA, or TFIIIC components (138, 131, 95) were fused to the two GAL4 domains. All combinations of fusion proteins with GAL4 DNA binding or activation domains were assayed. Most combinations of hybrid proteins gave background levels of -galactosidase activity. In contrast, when TFIIIB70 was fused to the GAL4 DNA binding domain and 131 to the GAL4 activation domain, high levels of -galactosidase activity were detected, suggesting that TFIIIB70 and 131 interact. The reciprocal assay gave similar results, whereas none of the fusion protein alone activated lacZ transcription ().

The 165 First Amino Acids of 131 Are Sufficient for Interaction with TFIIIB70

The interaction between TFIIIB70 and 131 was further characterized using deletion derivatives of 131. Some of the 131 mutants described in Fig. 2were fused to the activation domain of GAL4 and were tested for their interaction with TFIIIB70. We first investigated the role of the putative bHLH-Zip motif. As shown in Fig. 3, deletion of the basic or loop domain of the bHLH-Zip did not prevent 131 to interact with TFIIIB70. The -galactosidase activity measured with these mutant 131 fusions was only half that obtained with the wild type protein, indicating that the bHLH-Zip motif of 131 was not involved in TFIIIB70 binding. This conclusion was supported by the fact that the bHLH-Zip motif alone, when fused to the GAL4 activation domain, was unable to interact with TFIIIB70 (see Fig. 3).


Figure 3: Interaction of wild type or mutant 131 proteins with TFIIIB70. The two-hybrid system was used to study protein-protein interactions between 131 and TFIIIB70. The open reading frames of wild type or mutant 131 proteins were fused in frame with the GAL4 activation domain (GAL4 768-881). TFIIIB70 was fused in frame with the GAL4 DNA binding domain (GAL4 1-147). Transcription activation of the lacZ reporter gene was assayed by growing the transformed cells on selective medium and overlaying with X-gal agar. - and +, white and blue coloration of cell patches on X-gal plates, respectively. (-) indicates a slight blue coloration. -Galactosidase activity was measured at 30 °C for at least three independent transformants. Units are expressed in nanomoles of o-nitrophenyl--D-galactoside hydrolyzed per minute and per milligram of protein. Background level is around 1-4 units. ND, nondetermined.



To delineate the region of 131 required for interaction with TFIIIB70, we constructed a series of NH- or COOH-terminal fusions harboring the GAL4 DNA binding or activation domains. When 131-9TPR was fused to the activation domain of GAL4 and assayed with TFIIIB70 reciprocal fusion, high level of -galactosidase activity was measured. In contrast, background levels of -galactosidase activity were obtained with the N3 or N4 fusions, harboring the COOH-terminal moiety of 131. Even higher levels of -galactosidase activity were obtained with the smaller fusions 131-5TPR or 131-1TPR. In contrast, background -galactosidase activity was observed with only the NH-terminal acidic domain (131-0TPR). These results demonstrated that the NH-terminal 165 amino acids of 131, comprising the highly charged domain and the first TPR unit, were sufficient to interact with TFIIIB70 with the same efficiency as the entire 131 protein. This conclusion was supported by the fact that high levels of -galactosidase activity were obtained with the TPR2 and TPR3 mutant fusions whereas only background levels of -galactosidase activity were obtained with either the N2 or the TPR1 deleted 131 fusions. We checked by Western blotting analysis, using anti-GAL4 antibodies, that the fusion proteins were normally expressed (data not shown).

During these experiments, we noted that when fused to the GAL4 DNA binding domain, 131-5TPR and 131-1TPR were by themselves strong activators of pol II transcription. This was not the case for the entire 131 polypeptide ().

131 Interacts with the TFIIB-like Part of TFIIIB70

Next, we investigated interactions between 131 and deleted versions of TFIIIB70. The fragments of TFIIIB70 shown in Fig. 4were fused to the DNA binding or activation domains of GAL4 and assayed with the reciprocal 131 fusion. Not all combinations could be tested as we noted that when fused to the GAL4 DNA binding domain, the CTE moiety of TFIIIB70 was, by itself, a strong activator of pol II transcription, although this was not the case with the whole polypeptide (). As shown in , deletion of the zinc finger domain of TFIIIB70 (Zn) did not impair the interaction. Note that the zinc finger domain of TFIIB has been implicated in TFIIF binding (13) and as a target for glutamine-rich activation domains(32) . The CTE moiety of TFIIIB70 fused to the GAL4 activation domain did not detectably interact with 131. When the NH-terminal part of TFIIIB70 (CTE) was fused to the GAL4 activation domain, a small but significant level of -galactosidase activity was observed (), although the reciprocal combination gave background -galactosidase levels. When the zinc finger motif and the COOH-terminal part of TFIIIB70 were deleted (Zn/CTE), background -galactosidase activities were measured (), even though a light blue coloration was observed on plates (data not shown). Therefore, only the NH-terminal part of TFIIIB70 was able to weakly interact with 131, suggesting that the NH-terminal and COOH-terminal moiety cooperated for optimal interaction.

We reasoned that wild type levels of interaction could be restored by using truncated versions of 131. Thus, the Zn, CTE, and Zn/CTE deleted TFIIIB70 proteins, fused to the GAL4 DNA binding domain, were assayed with the 131-9TPR, 131-5TPR, 131-1TPR, and 131-0TPR reciprocal fusions described in Fig. 3. With or without the zinc finger motif, TFIIIB70 interacted with the 165 first amino acids of 131 (). When TFIIIB70-CTE or TFIIIB70-Zn/CTE deleted proteins were assayed, the highest level of interaction was observed with the 131-5TPR hybrid. Low but still significant levels of interaction were observed with the 131-9TPR or 131-1TPR hybrids, whereas background -galactosidase activity was found with 131-0TPR. These results clearly demonstrated that the NH-terminal part of 131 interacts with the TFIIB-like moiety of TFIIIB70.


DISCUSSION

Using a yeast interaction system, we have mapped regions of TFIIIB and TFIIIC that interact within the TFIIIBTFIIICDNA complex. We found that the NH-terminal domain of TFIIIB70, that is structurally similar to TFIIB, interacts with 131 subunit of TFIIIC, whereas the COOH-terminal half binds to TBP. The 131 interacting zone was found to be confined to its NH-terminal end. The results support the model in which TFIIIB70 bridges the pol III initiation complex through its interactions with TBP, 131, and C34 subunit of pol III (this work, Refs. 6-8 and 33).

A direct interaction between TBP and TFIIIB70 was previously reported (7, 33). We found here that TBP interacts with the CTE moiety of TFIIIB70, whereas no binding to the TFIIB-like domain could be detected. In contrast, Khoo et al.(33) recently found by affinity chromatography, using glutathione S-transferase fusions with wild type or deleted forms of TFIIIB70, that S-labeled TBP bound to both the CTE and the TFIIB-like domains of TFIIIB70. The discrepancy with our own results may be due to an improper folding of the TFIIB-like domain on the membrane preventing TBP recognition. However, the interaction between TBP and the CTE of TFIIIB70 could also be observed using the two-hybrid system, whereas again no interaction was detected with the TFIIB-like part.()Alternatively, the large excess of immobilized TFIIIB70-GST fusion protein may have favored some nonspecific binding of labeled TBP to the TFIIB homologous region. This possibility was suggested by the fact that the full-length TBP probe was specifically retained only by the carboxyl-terminal domain of TFIIIB70, residues 263-596, and that mutations in TBP specifically affected its interaction with the CTE moiety(33) . Therefore, the presence of two distinct TBP-interacting regions in TFIIIB70 remains an open question. We found that the integrity of the CTE, but not of the TFIIB-like domain, was important for TBP binding. Previous studies have indicated that deletions of NH- or COOH-terminal residues of TFIIIB70 disrupt its function in vivo(20, 21) . Since a deletion of 140 residues at the NH-terminal end of TFIIIB70 did not impair TBP binding in vitro (Fig. 1), it seems likely that the lethality of yeast strains bearing a derivative of TFIIIB70 deleted of 40 residues at the NH-terminal end (21) is not a consequence of a TFIIIB70TBP interaction defect. We found, instead, that this TFIIB-like domain was involved in 131 binding.

The protein domains of 131 and TFIIIB70 involved in their interaction were mapped. Our results demonstrated that 165 residues at the NH-terminal end of 131 were sufficient to interact with TFIIIB70 as efficiently as the entire protein. These are in vivo interactions. Formally, there is the possibility that 131 and TFIIIB70 interact indirectly via a third, intervening component (i.e. another subunit of TFIIIC). However, the possibility of the stable assembly of TFIIIC harboring only 165 residues of 131 appears very unlikely, especially since we found that many small deletions throughout 131 were lethal (Fig. 2). In addition, we detected no interaction of TFIIIB70 with the 95 subunit that cross-link next to 131 in TFIIICDNA complexes(25, 26) . While this paper was in preparation, Khoo et al.(33) reported that each, the NH-terminal, middle, and COOH-terminal thirds of 131, bound to TFIIIB70 independently. We did not detect an interaction between the COOH-terminal part of 131 and TFIIIB70 (see Fig. 3), but our results showed that the COOH-terminal part of 131 (past TPR5) interfered with the interaction between the NH-terminal part of 131 and the TFIIB-like moiety of TFIIIB70 (). This suggests the existence of intramolecular interactions, within 131, that shield the TFIIIB70 interaction domain. This contention was supported by our observation that the strong pol II activator properties of 131-5TPR or 131-1TPR fused to GAL4 DNA binding domain were masked within the entire 131 polypeptide. 131-9TPR that interacted less efficiently with the TFIIB-like part of TFIIIB70 also lacked activator property (see Tables I and II). Therefore, intramolecular interactions in 131 may possibly involve the two blocks of TPR, TPR1-5, and TPR6-9.

Our results demonstrated an interaction between the NH-terminal parts of TFIIIB70 and 131, but did not exclude a role for the COOH-terminal part of TFIIIB70. In fact, the COOH-terminal part of TFIIIB70 may play an active role in the establishment of the interaction between TFIIIB70 and 131. Indeed, the presence of the CTE was important for interaction with 131, 131-9TPR, or 131-1TPR, whereas it was dispensable for interaction with the 131-5TPR fusion (). One could imagine, for example, that the COOH-terminal part of TFIIIB70 enhances 131TFIIIB70 interaction by modifying the conformation of 131, allowing the interaction between the NH-terminal parts of TFIIIB70 and 131. As TBP binds to the CTE of TFIIIB70, another explanation could be that the binding of TBP to the CTE is necessary for the interaction of the TFIIB-like part of TFIIIB70 with 131. This hypothesis is supported by the observation that TBP stabilizes TFIIIB70TFIIICDNA complexes(6) .

As in the case of 131, a subdomain of TFIIIB70, the CTE, displayed strong pol II activator properties that were repressed in the entire protein. This observation may or may not be related to the pol III activation role of TFIIIB70. At least, the masking of this activation domain in the entire polypeptide strongly suggests the existence of intramolecular interactions between the TFIIB-like part of TFIIIB70 and the CTE. We wondered if the strong pol II activator properties observed with the CTE or some particular 131 fusions could be due to a direct interaction with TFIIB (we have seen that 131-5TPR or 131-1TPR interacted strongly with the TFIIB-like part of TFIIIB70). However, no interaction could be detected between TFIIB fused to the GAL4 DNA binding domain and the reciprocal 131-5TPR, 131-1TPR, or CTE fusions. Despite their sequence homologies, TFIIB and the TFIIB-like part of TFIIIB70 were not functionally exchangeable.()

The present interaction studies extend the functional similarities, previously noted(20, 21, 22) , between TFIIB and TFIIIB70. The role of 131 appears closely related to the function of class II transactivators that bind to TFIIB (10, 11, 12) and, as shown recently (34), induce a conformation change in the factor molecule probably essential for the subsequent steps of preinitiation complex assembly.

  
Table: Intrinsic activation properties of DNA binding fusions proteins

The symbols + and - are attributed to blue coloration and no coloration on X-gal plates, respectively, when the TFIIIB70 or 131 derivatives, fused to the DNA binding domain, were assayed with no counterpart.


  
Table: Interaction of wild type or mutant TFIIIB70 proteins with 131 or COOH-terminal truncated 131

The two-hybrid system was used to study protein-protein interactions between 131 and TFIIIB70. Wild type or deleted derivatives of TFIIIB70, fused in frame with the GAL4 DNA binding domain, were assayed with the reciprocal 131 fusions. Explanations of the panel are the same as in Fig. 3. ND, nondetermined. -Galactosidase units in parentheses indicate reciprocal combinations, with 131 fused to the GAL4 DNA binding domain.



FOOTNOTES

*
This work was supported by the Biotechnology Programme Grant BIO2-CT92-0090 from the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Genetics Dept. SK-50, University of Washington, Seattle, WA 98195.

To whom correspondence and reprint requests should be addressed. Tel.: 33-1-69082236; Fax: 33-1-69084712.

The abbreviations used are: TBP, TATA-binding protein; pol III, RNA polymerase III; TPR, tetratricopeptide repeat; bHLH-Zip, basic-helix-loop-helix-zipper; CTE, carboxyl-terminal extension; X-gal, 5-bromo-4-chloro-3-indoyl -D-galactoside; PAGE, polyacrylamide gel electrophoresis.

P. Bartel and S. Fields, personal communication.

G. Dieci, personal communication.

S. Shaaban, unpublished results.

C. Conesa, unpublished results.


ACKNOWLEDGEMENTS

We greatly thank Christian Marck who initiated the mutagenesis analysis, Olivier Lefebvre and Laurence Damier for assistance with the two hybrid experiments, Jochen Rüth for the gift of pAS-TBP and pACTII-JR and for help with the suppressor analysis, and Janine Huet for her contribution to the FarWestern analysis. We acknowledge Stephen Elledge for providing us with the two hybrid strains and plasmids, James Hopper and Colleen Lebo for providing the anti-GAL4 antibodies, and Michael Hampsey for the SUA7 plasmids.


REFERENCES
  1. Gabrielsen, O. S., and Sentenac, A.(1991) Trends Biochem. Sci.16, 412-416 [CrossRef][Medline] [Order article via Infotrieve]
  2. Geiduschek, E. P., and Kassavetis, G. A.(1992) Transcriptional Regulation, pp. 247-280, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  3. Willis, I. M.(1993) Eur. J. Biochem.212, 1-11 [Abstract]
  4. White, R. J.(1994) in RNA Polymerase III Transcription, Molecular Biology Intelligence Unit, R. G. Landes Co., Austin, TX
  5. Kassavetis, G. A., Braun, B. R., Nguyen, L. H., and Geiduschek, E. P. (1990) Cell60, 235-245 [Medline] [Order article via Infotrieve]
  6. Kassavetis, G. A., Joazeiro, C. A. P., Pisano, M., Geiduschek, E. P., Colbert, T., Hahn, S., and Blanco, J. A.(1992) Cell71, 1055-1064 [Medline] [Order article via Infotrieve]
  7. Huet, J., Conesa, C., Manaud, N., Chaussivert, N., and Sentenac, A. (1994) Nucleic Acids Res.22, 3433-3439 [Abstract]
  8. Werner, M., Chaussivert, N., Willis, I. A., and Sentenac, A.(1993) J. Biol. Chem.268, 20721-20724 [Abstract/Free Full Text]
  9. Buratowski, S., Hahn, S., Guarente, L., and Sharp, P. A.(1989) Cell56, 549-561 [Medline] [Order article via Infotrieve]
  10. Lin, Y. S., and Green, M.(1991) Cell64, 971-981 [Medline] [Order article via Infotrieve]
  11. Lin, Y.-S., Ha, I., Maldonado, E., Reinberg, D., and Green, M.(1991) Nature353, 569-571 [CrossRef][Medline] [Order article via Infotrieve]
  12. Roberts, S. G. E., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R.(1993) Nature363, 741-744 [CrossRef][Medline] [Order article via Infotrieve]
  13. Ha, I., Roberts, S., Maldonado, E., Sun, X., Kim, L. U., Green, M. R., and Reinberg, D.(1993) Genes & Dev.7, 1021-1032
  14. Barberis, A., Müller, C. W., Harrison, S. C., and Ptashne, M. (1993) Proc. Natl. Acad. Sci. U. S. A.90, 5628-5632 [Abstract]
  15. Buratowski, S., and Zhou, H.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 5633-5637 [Abstract]
  16. Hisatake, K., Roeder, R. G., and Horikoshi, M.(1993) Nature363, 744-747 [CrossRef][Medline] [Order article via Infotrieve]
  17. Malik, S., Hisatake, K., Sumimoto, H., Horikoshi, M., and Roeder, R. G. (1991) Proc. Natl. Acad. Sci. U. S. A.88, 9553-9557 [Abstract]
  18. Ha, I., Lane, W. S., and Reinberg, D.(1991) Nature352, 689-695 [CrossRef][Medline] [Order article via Infotrieve]
  19. Pinto, I., Ware, D. E., and Hampsey, M.(1992) Cell68, 977-988 [Medline] [Order article via Infotrieve]
  20. Buratowski, S., and Zhou, H.(1992) Cell71, 221-230 [Medline] [Order article via Infotrieve]
  21. Colbert, T., and Hahn, S.(1992) Genes & Dev.6, 1940-1949
  22. López-De-León, A., Librizzi, M., Puglia, K., and Willis, I. (1992) Cell71, 211-220 [Medline] [Order article via Infotrieve]
  23. Marck, C., Lefebvre, O., Carles, C., Riva, M., Chaussivert, N., Ruet, A., and Sentenac, A.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 4027-4031 [Abstract]
  24. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) Cell75, 805-816 [Medline] [Order article via Infotrieve]
  25. Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J.9, 2197-2205 [Abstract]
  26. Bartholomew, B., Kassavetis, G. A., and Geiduschek, E. P.(1991) Mol. Cell. Biol.11, 5181-5189 [Medline] [Order article via Infotrieve]
  27. Braun, B. R., Bartholomew, B., Kassavetis, G. A., and Geiduschek, E. P. (1992) J. Mol. Biol.228, 1063-1077 [Medline] [Order article via Infotrieve]
  28. Rameau, R., Puglia, K., Crowe, A., Sethy, I., and Willis, I.(1994) Mol. Cell. Biol.14, 822-830 [Abstract]
  29. Lefebvre, O., Rüth, J., and Sentenac, A.(1994) J. Biol. Chem.269, 23374-23381 [Abstract/Free Full Text]
  30. Fields, S., and Song, O. K.(1989) Nature340, 245-246 [CrossRef][Medline] [Order article via Infotrieve]
  31. Chien, C. T., Bartel, P. L., Sternglanz, R., and Fields, S.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 9578-9582 [Abstract]
  32. Colgan, J., Wampler, S., and Manley, J. M.(1993) Nature362, 549-553 [CrossRef][Medline] [Order article via Infotrieve]
  33. Khoo, B., Brophy, B., and Jackson, S. P.(1994) Genes & Dev.8, 2879-2890
  34. Roberts, S. G. E., and Green, M. R.(1994) Nature371, 717-720 [CrossRef][Medline] [Order article via Infotrieve]

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