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Molecular and Cellular Biology, December 2002, p. 8735-8743, Vol. 22, No. 24
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.24.8735-8743.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Interdependent Interactions between TFIIB, TATA Binding Protein, and DNA

Robin M. Buratowski, Jessica Downs,{dagger} and Stephen Buratowski*

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Received 29 May 2002/ Returned for modification 18 July 2002/ Accepted 24 September 2002

Temperature-sensitive mutants of TFIIB that are defective for essential interactions were isolated. One mutation (G204D) results in disruption of a protein-protein contact between TFIIB and TATA binding protein (TBP), while the other (K272I) disrupts an interaction between TFIIB and DNA. The TBP gene was mutagenized, and alleles that suppress the slow-growth phenotypes of the TFIIB mutants were isolated. TFIIB with the G204D mutation [TFIIB(G204D)] was suppressed by hydrophobic substitutions at lysine 239 of TBP. These changes led to increased affinity between TBP and TFIIB. TFIIB(K272I) was weakly suppressed by TBP mutants in which K239 was changed to hydrophobic residues. However, this mutant TFIIB was strongly suppressed by conservative substitutions in the DNA binding surface of TBP. Biochemical characterization showed that these TBP mutants had increased affinity for a TATA element. The TBPs with increased affinity could not suppress TFIIB(G204D), leading us to propose a two-step model for the interaction between TFIIB and the TBP-DNA complex.

* Corresponding author. Mailing address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Bldg. C1, Room 214, 240 Longwood Ave., Boston, MA 02115. Phone: (617) 432-0696. Fax: (617) 738-0516. E-mail: steveb{at}hms.harvard.edu.

{dagger} Present address: Department of Biochemistry, Cambridge University, Cambridge, United Kingdom.

Molecular and Cellular Biology, December 2002, p. 8735-8743, Vol. 22, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.24.8735-8743.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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