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Molecular and Cellular Biology, December 2002, p. 8744-8755, Vol. 22, No. 24
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.24.8744-8755.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A TATA Binding Protein Mutant with Increased Affinity for DNA Directs Transcription from a Reversed TATA Sequence In Vivo

J. Vaughn Spencer and Karen M. Arndt^*

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Received 21 May 2002/ Returned for modification 18 July 2002/ Accepted 24 September 2002

The TATA-binding protein (TBP) nucleates the assembly and determines the position of the preinitiation complex at RNA polymerase II-transcribed genes. We investigated the importance of two conserved residues on the DNA binding surface of Saccharomyces cerevisiae TBP to DNA binding and sequence discrimination. Because they define a significant break in the twofold symmetry of the TBP-TATA interface, Ala100 and Pro191 have been proposed to be key determinants of TBP binding orientation and transcription directionality. In contrast to previous predictions, we found that substitution of an alanine for Pro191 did not allow recognition of a reversed TATA box in vivo; however, the reciprocal change, Ala100 to proline, resulted in efficient utilization of this and other variant TATA sequences. In vitro assays demonstrated that TBP mutants with the A100P and P191A substitutions have increased and decreased affinity for DNA, respectively. The TATA binding defect of TBP with the P191A mutation could be intragenically suppressed by the A100P substitution. Our results suggest that Ala100 and Pro191 are important for DNA binding and sequence recognition by TBP, that the naturally occurring asymmetry of Ala100 and Pro191 is not essential for function, and that a single amino acid change in TBP can lead to elevated DNA binding affinity and recognition of a reversed TATA sequence.

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* Corresponding author. Mailing address: Department of Biological Sciences, 269 Crawford Hall, 4249 Fifth Ave., University of Pittsburgh, Pittsburgh, PA 15260. Phone: (412) 624-6963. Fax: (412) 624-4759. E-mail: arndt{at}pitt.edu.

Present address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.

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Molecular and Cellular Biology, December 2002, p. 8744-8755, Vol. 22, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.24.8744-8755.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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