Functional dissection of the B'' component of RNA polymerase III transcription factor IIIB: A scaffolding protein with multiple roles in assembly and initiation of transcription

Transcription factor IIIB (TFIIIB), the central transcription factor of Saccharomyces cerevisiae RNA polymerase III, is composed of TATA-binding protein, the TFIIB-related protein Brf, and B '', B '', the last component to enter the TFIIIB-DNA complex, confers extremely tight DNA binding on TFIIIB, Terminally and internally deleted B '' derivatives were tested for competence to form TFIIIB-DNA complexes by TFIIC-dependent and -independent pathways on the SUP4 tRNA(Tyr) and U6 snRNA (SNR6) genes, respectively, and for transcription. Selected TFIIIB-TFIIIC-DNA complexes assembled with truncated B '' were analyzed by DNase I footprinting, and the surface topography of B '' in the TFIIIB-DNA complex was also analyzed by hydroxyl radical protein footprinting, These analyses define functional domains of B '' and also reveal roles in start site selection by RNA polymerase III and in clearing TFIIIC from the transcriptional start. Although absolutely required for transcription, B '' can be extensively truncated, Core proteins retaining as few as 176 (of 594) amino acids remain competent to transcribe the SNR6 gene in vitro, TFIIIC-dependent assembly on DNA and transcription requires a larger core of B '': two domains (I and II) that are required for SNR6 transcription on an either-or basis are simultaneously required for TFIIIC-dependent assembly of DNA complexes and transcription. Domains I and II of B '' are buried upon assembly of the TFIIIB-DNA complex, as determined by protein footprinting, The picture of the TFIIIB-DNA complex that emerges is that B '' serves as its scaffold and is folded over in the complex so that domains I and II are near one another.