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Molecular and Cellular Biology, September 1998, p. 5587-5599, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Functional and Structural Organization of Brf, the TFIIB-Related Component of the RNA Polymerase III Transcription Initiation Complex

George A. Kassavetis,* Ashok Kumar,* Enrique Ramirez, and E. Peter Geiduschek

Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634

Received 23 April 1998/Returned for modification 26 May 1998/Accepted 3 June 1998

Brf is the TFIIB-related component of Saccharomyces 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.

* Corresponding author. Mailing address: Department of Biology and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0634. Phone: (619) 534-2451. Fax: (619) 534-7073. E-mail for George A. Kassavetis: gak{at}ucsd.edu. E-mail for Ashok Kumar: akumar{at}biomail.ucsd.edu.

Molecular and Cellular Biology, September 1998, p. 5587-5599, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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