Transcription by RNA polymerase III involves the formation of a preinitiation complex by template-bound transcription factors, and the selective recruitment of the enzyme. The modalities of Pol III recruitment are still poorly understood. At least two subunits of Pol III (C34 and C31) are involved in Pol III recruitment and/or RNA chain initiation (8
). C34 interacts with TFIIIB70, one component of TFIIIB, and this interaction plays a critical role in enzyme recruitment and open complex formation (8
). Here we have characterized a novel subunit of yeast Pol III, C17, and shown by protein-protein interaction assays that it may contribute to the specific recognition of TFIIIB. The essentiality of C17 suggests that its role is not redundant with that of C34.
The possibility that C17 was another subunit of Pol III had not been seriously considered because of its erratic electrophoretic migration (slower or faster than AC19) and of its high stoichiometry, close to 2, based on Coomassie blue staining (Fig. ). Furthermore, the identification of C17 as a close homolog to a mammalian hormone receptor (37
) was not very encouraging. Nevertheless, the data obtained in vivo and in vitro convincingly demonstrate that C17 is a bona fide subunit of Pol III: first, C17 is essential for cell viability, like all the other Pol III subunits; extinction of RPC17
gene expression in vivo causes a strong defect in tRNA synthesis; and finally, C17 was found to interact with at least two Pol III subunits and with a critical component of TFIIIB. In comparison to other specific subunits of Pol III (unrelated to Pol I and Pol II subunits), it is striking that the level of sequence conversion from yeast to human is higher in the case of C17 than in the case of C82 or C31. It is therefore likely that the mammalian proteins homologous to C17 belong to Pol III, which seems at odds with their proposed role as a hormone receptor. The functional replacement of the RPC17
gene by the human gene would deserve to be attempted, although the mammalian homolog of C53 (27
), C34 (J. C. Andrau and I. Brun, unpublished results) or C11 (12
) subunits could not replace their yeast counterparts.
The in vivo extinction of RPC17
gene expression caused a strong decrease of the de novo synthesis of tRNA that led to a significant fall in the steady-state amounts of tRNA (from 35 to 70% after 6 h or 24 h of antibiotic treatment, respectively). A threefold drop in tRNA levels following C17 gene shutoff was accompanied by a fivefold decline in the cell growth rate. This observation correlates well with a model where the regulation of Pol III transcription would provide a mechanism for regulating the cell growth rate (reference 54
and references therein).
Protein-protein interaction assays showed that C17 binds specifically to at least two components of the Pol III transcription apparatus that play a critical role in chain initiation, namely, C31 on the polymerase side and TFIIIB70. These data support a model in which C17 contributes to the productive positioning of the Pol III by TFIIIB on class III promoters. All evidence garnered to date pointed to C34-TFIIIB70 interaction as the major determinant in Pol III recruitment. Of eight Pol III subunits that could be cross-linked on SUP4
promoter DNA in initiation complexes, C34 extended the furthest upstream, from bp +6 to −17 (5
). C34 was found to interact in vitro and in vivo with TFIIIB70 (34
), and mutations in C34 decreasing its interaction with TFIIIB70 impaired Pol III recruitment (a defect that could be compensated in vitro by increasing the concentration of the mutant enzyme) or, more unexpectedly, affected the ability of Pol III to form open complexes (8
). A proper C34-TFIIIB70 interaction was suggested to be important to trigger the isomerization step required to shift the enzyme into an initiation-competent configuration (8
). Interestingly, TFIIIB-DNA complexes assembled with certain truncated versions of TFIIIB70 or B" were found to recruit transcriptionally inactive forms of Pol III unable to initiate transcription (31
). These observations underscored the role of TFIIIB-polymerase interactions at postrecruitment steps of transcription initiation (31
). C31 is another candidate for participating in the Pol III recruitment/activation step. This polypeptide is part of a subcomplex of three subunits, C82, C34, and C31, conserved in human Pol III, that dissociates from the enzyme under adverse conditions and that is required for initiation (52
). A Pol III mutant enzyme containing a truncated form of C31 was shown to be specifically deficient in chain initiation, suggesting a defect in TFIIIB interaction or in open complex formation (50
). C82, which belongs to the labile subcomplex, may also be involved in transcription initiation. It was unexpected, therefore, to discover an additional Pol III-specific subunit, C17, that contributes to TFIIIB recognition. The observation that several Pol III-specific subunits appear to be involved in enzyme recruitment and chain initiation strongly suggests that multiple contacts with the preinitiation complex are needed to position the enzyme properly at the start site and to set off the complex process of RNA chain initiation.
C17 being essential for growth, its role cannot be redundant with that of C34 or the other specific subunits. Protein-protein interaction assays indicate that C17 provides another grip on TFIIIB by interacting with the TFIIB-related part of TFIIIB70. GST pull-down experiments showed that C34 also binds to the N-terminal half of TFIIIB70 (34
). The interaction of both Pol III-specific subunits, C34 and C17, with the TFIIB-like part of TFIIIB70 accounts well for the major role of this TFIIIB domain in Pol III recruitment. Indeed, the N-terminal half of TFIIIB70 (residues 1 to 282) was shown to retain nearly full transcriptional activity in vitro in a TFIIIC-independent assay using the yeast SNR6
). The interaction domains on TFIIIB70 for C34 and C17 remain to be mapped precisely. It is known that C34 interacts with the direct repeat region, not with the zinc-binding domain (34
). Mutagenesis of TFIIIB70 also revealed C-terminal residues critical for interaction with C34 and TBP (2
). Our coimmunoprecipitation experiments showed that a TFIIIB70 fragment lacking ≈20 kDa at its N terminus interacted with C17 as efficiently as the full-length protein. This observation suggested that the C17-binding domain lay C terminal of the first imperfect repeat, within the TFIIB-like half of TFIIIB70.
As mentioned above, the role of C34 and C17 is unlikely to be redundant since both are essential components. Pol III-DNA cross-linking experiments using 4-S-dTMP as the “zero distance” photo-cross-linking agent revealed a direct contact of several Pol III-specific subunits with DNA: C82, C34, and C31, a triad of subunits involved in chain initiation (5
). Two unidentified small subunits (18 to 14 kDa) were also found to contact DNA in ternary transcription complexes. Therefore, there is the possibility that C17, like C34, contacts both TFIIIB70 and DNA in the transcription complex. A more precise mapping and mutagenesis of C17-TFIIIB70 interaction domains will hopefully reveal the role of this interaction in Pol III recruitment, start site selection, and/or some subsequent steps leading to RNA chain initiation.