CONSERVED FUNCTIONAL DOMAINS OF THE RNA-POLYMERASE-III GENERAL TRANSCRIPTION FACTOR BRF

In Saccharomyces cerevisiae, two components of the RNA polymerase III (Pol III) general transcription factor TFIIIB are the TATA-binding pro tein (TBP) and the B-related factor (BRF), so called because its amino -terminal half is homologous to the Pol II transcription factor IIB (T FIIB). We have cloned BRF genes from the yeasts Kluyveromyces lactis a nd Candida albicans, Despite the large evolutionary distance between t hese species and S. cerevisiae, the BRF proteins are conserved highly. Although the homology is most pronounced in the amino-terminal half, conserved regions also exist in the carboxy-terminal half that is uniq ue to BRF. By assaying for interactions between BRF and other Pol III transcription factors, we show that it is able to bind to the 135-kD s ubunit of TFIIIC and also to TBP. Surprisingly, in addition to binding the TFIIB-homologous amino-terminal portion of BRF, TBP also interact s strongly with the carboxy-terminal half. Deleting two conserved regi ons in the BRF carboxy-terminal region abrogates this interaction. fur thermore, TBP mutations that selectively inhibit Pol III transcription in vivo impair interactions between TBP and the BRF carboxy-terminal domain. Finally, we demonstrate that BRF but not TFIIB binds the Pol I II subunit C34 and we define a region of C34 necessary for this intera ction. These observations provide insights into the roles performed by BRF in Pol III transcription complex assembly.