Open AccessFunctional and Structural Organization of Brf, the TFIIB-Related Component of the RNA Polymerase III Transcription Initiation Complex
Author(s) -
George A. Kassavetis,
Ashok Kumar,
Enrique Ramírez,
E. Peter Geiduschek
Publication year - 1998
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.18.9.5587
Subject(s) - transcription factor ii a , biology , transcription (linguistics) , rna polymerase iii , dna , transcription factor ii b , genetics , computational biology , transcription preinitiation complex , transcription factor , polymerase , microbiology and biotechnology , promoter , gene , gene expression , rna dependent rna polymerase , linguistics , philosophy
Brf is the TFIIB-related component ofSaccharomyces cerevisiae RNA polymerase III transcription initiation factor IIIB (TFIIIB). An extensive set of Brf fragments has been examined for the abilities to assemble the TFIIIB-DNA complex and recruit RNA polymerase III to accurately initiate transcription. The principal TFIIIB-assembly function of Brf was found to be contributed by a C-proximal segment spanning amino acids 435 to 545, while the principal transcription-directing function was contributed by a segment of its N-proximal, TFIIB-homologous half. The diverse activities of Brf were also reconstituted from combined fragments. The fragments spanning amino acids 1 to 282 and 284 to 596 were found to assemble into TFIIIB-DNA and TFIIIC-TFIIIB-DNA complexes that were very stable, transcriptionally highly active, and indistinguishable (by in vitro footprinting) from complexes formed with intact Brf. The proximities of the individual halves of split Brf to DNA were extensively mapped by photochemical cross-linking of the TFIIIB-DNA complex. We also identified sites of interaction of Brf fragments with TATA-binding protein (TBP), taking advantage of a recently completed mutational analysis of the TBP surface. The constraints established by these analyses specify a global model of the functional segments of Brf and how they fit into the structure of the TFIIIB-DNA complex.