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 TFIIIC-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.
Kumar
A
A
Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0634, USA.
Kassavetis
G A
GA
Geiduschek
E P
EP
Hambalko
M
M
Brent
C J
CJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D015342:DNA Probes / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D017665:Hydroxyl Radical / Q000737:chemistry; D046911:Macromolecular Substances; D015394:Molecular Structure; D012320:RNA Polymerase III / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D015342:DNA Probes; D004271:DNA, Fungal; D046911:Macromolecular Substances; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D017665:Hydroxyl Radical; D012320:RNA Polymerase III