The role of transcription initiation factor IIIB subunits in promoter opening probed by photochemical cross-linking.
The core transcription initiation factor (TF) IIIB recruits its conjugate RNA polymerase (pol) III to the promoter and also plays an essential role in promoter opening. TFIIIB assembled with certain deletion mutants of its Brf1 and Bdp1 subunits is competent in pol III recruitment, but the resulting preinitiation complex does not open the promoter. Whether Brf1 and Bdp1 participate in opening the promoter by direct DNA interaction (as sigma subunits of bacterial RNA polymerases do) or indirectly by their action on pol III has been approached by site-specific photochemical protein-DNA cross-linking of TFIIIB-pol III-U6 RNA gene promoter complexes. Brf1, Bdp1, and several pol III subunits can be cross-linked to the nontranscribed strand of the U6 promoter at base pair -9/-8 and +2/+3 (relative to the transcriptional start as +1), respectively the upstream and downstream ends of the DNA segment that opens up into the transcription bubble. Cross-linking of Bdp1 and Brf1 is detected at 0 degrees C in closed preinitiation complexes and at 30 degrees C in complexes that are partly open, but also it is detected in mutant TFIIIB-pol III-DNA complexes that are unable to open the promoter. In contrast, promoter opening-defective TFIIIB mutants generate significant changes of cross-linking of polymerase subunits. The weight of this evidence argues in favor of an indirect mode of action of TFIIIB in promoter opening.
Kassavetis
George A
GA
Division of Biological Sciences and the Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634, USA. gak@uscd.edu
Han
Shulin
S
Naji
Souad
S
Geiduschek
E Peter
EP
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D035361:Transcription Factor TFIIIB / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic
C090956:BRF1 protein, S cerevisiae; C474805:Bdp1 protein, S cerevisiae; D003432:Cross-Linking Reagents; D029701:Saccharomyces cerevisiae Proteins; D035361:Transcription Factor TFIIIB; D004247:DNA