Complex interactions between yeast TFIIIB and TFIIIC.

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 tau) via its tau 131 subunit. The protein domains of tau 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.

Directing transcription of an RNA polymerase III gene via GAL4 sites.

A yeast chimeric RNA polymerase III transcription system was constructed to explore the ordered, multistep process of gene activation in vivo. A promoter-deficient U6 RNA gene harboring GAL4-binding sites could be reactivated by fusing the GAL4 DNA-binding domain to components of the general transcription factor TFIIIC (tau) or TFIIIB. Expression of chimeric tau 138 or tau 131 (but not tau 95) subunits activated transcription from GAL4-binding sites located at various positions, including upstream of or within the gene. The function(s) of the B block binding domain of TFIIIC was provided by the fused GAL4-(1-147) domain. The GAL4-(1-147)-TFIIIB70 fusion protein acted at a distance like an activator of transcription. In contrast, none of the 10 different GAL4-(1-147)-polymerase subunit fusions was able to induce transcription, suggesting that RNA polymerase recruitment is not sufficient to initiate transcription.

Interactions between yeast TFIIIB components.

Yeast transcription factor TFIIIB is a multicomponent factor comprised of the TATA-binding protein TBP and of associated factors TFIIIB70 and B". Epitope-tagged or histidine-tagged TFIIIB70 could be quantitatively removed from TFIIIB by affinity chromatography. TBP and B" (apparent mass 160-200 kDa) could be easily separated by gel filtration or ion-exchange chromatography. While only weak interactions were detected between TBP and B", direct binding of [35S]-labeled TBP to membrane-bound TFIIIB70 could be demonstrated in absence of DNA. On tRNA genes, there was no basal level of transcription in the complete absence of TBP. The two characterized TFIIIB components (recombinant rTFIIIB70 and rTBP) and a fraction cochromatographing with B" activity were found to be required for TFIIIC-independent transcription of the TATA-containing U6 RNA gene in vitro. Therefore, beside the TFIIIC-dependent assembly process, each TFIIIB component must have an essential role in DNA binding or RNA polymerase recruitment.

Interactions between yeast TFIIIB components.

Yeast transcription factor TFIIIB is a multicomponent factor comprised of the TATA-binding protein TBP and of associated factors TFIIIB70 and B". Epitope-tagged or histidine-tagged TFIIIB70 could be quantitatively removed from TFIIIB by affinity chromatography. TBP and B" (apparent mass 160-200 kDa) could be easily separated by gel filtration or ion-exchange chromatography. While only weak interactions were detected between TBP and B", direct binding of [35S]-labeled TBP to membrane-bound TFIIIB70 could be demonstrated in absence of DNA. On tRNA genes, there was no basal level of transcription in the complete absence of TBP. The two characterized TFIIIB components (recombinant rTFIIIB70 and rTBP) and a fraction cochromatographing with B" activity were found to be required for TFIIIC-independent transcription of the TATA-containing U6 RNA gene in vitro. Therefore, beside the TFIIIC-dependent assembly process, each TFIIIB component must have an essential role in DNA binding or RNA polymerase recruitment.

Interaction between a complex of RNA polymerase III subunits and the 70-kDa component of transcription factor IIIB.

A system that detects the formation of complexes between different proteins by linking them to separate domains of the GAL4 transcription activator protein has been used to study protein-protein interactions between four essential and unique subunits of yeast RNA polymerase III (C82, C53, C34 and C31), the 70-kDa component of the initiation transcription factor IIIB (TFIIIB70) and the TATA-binding protein. We found that C82, C34, and C31 are able to combine with each other in vivo and that C34 interacts with TFIIIB70. These results suggest that C34 and TFIIIB70 are specificity determinants of the RNA polymerase III-TFIIIB interaction.

The TFIIIB-assembling subunit of yeast transcription factor TFIIIC has both tetratricopeptide repeats and basic helix-loop-helix motifs.

The multisubunit yeast transcription factor IIIC (TFIIIC; also called tau) can undergo considerable conformational changes upon binding to the A and B blocks of tRNA genes. After binding to DNA encoding tRNA (tDNA), TFIIIC acts as an assembly factor to recruit an initiation factor, TFIIIB, via its tau 131 subunit. We have cloned the gene encoding the tau 131 subunit and named it TFC4. This gene is unique, essential for cell viability, and encodes a M(r) 120,153 protein. Epitope-tagging and mobility-shift assays indicated the presence of a single tau 131 subunit in TFIIIC-tDNA complexes. tau 131 contains two sequence motifs, accounting for nearly one-half of the protein mass, that may provide a molecular explanation for the properties of TFIIIC-tDNA complex. A series of 11 copies of the tetratricopeptide repeat motif may account for the flexibility and interaction properties of TFIIIC. A motif akin to the basic helix-loop-helix motif of MyoD suggests the direct involvement of tau 131 in promoting DNA binding of TFIIIB.