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Mol Cell Biol, March 1998, p. 1682-1691, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Architecture of Protein and DNA Contacts within the TFIIIB-DNA Complex

Trenton Colbert, Sally Lee,dagger Greg Schimmack, and Steven Hahn*

Howard Hughes Medical Institute and Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024

Received 30 September 1997/Returned for modification 17 November 1997/Accepted 24 November 1997

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.

* Corresponding author. Mailing address: Howard Hughes Medical Institute and Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Mail stop A1-162, Seattle, WA 98109-1024. Phone: (206) 667-5261. Fax: (206) 667-6497. E-mail: shahn{at}fhcrc.org.

dagger Present address: Incyte Pharmaceuticals, Palo Alto, CA 94086.




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