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Molecular and Cellular Biology, April 2000, p. 2385-2399, Vol. 20, No. 7
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Impaired Core Promoter Recognition Caused by Novel Yeast TAF145 Mutations Can Be Restored by Creating a Canonical TATA Element within the Promoter Region of the TUB2 Gene

Yoshihiro Tsukihashi, Tsuyoshi Miyake,dagger Masashi Kawaichi, and Tetsuro Kokubo*

Division of Gene Function in Animals, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan

Received 28 September 1999/Returned for modification 29 November 1999/Accepted 10 January 2000

The general transcription factor TFIID, which is composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs), has been shown to play a crucial role in recognition of the core promoters of eukaryotic genes. We isolated Saccharomyces cerevisiae yeast TAF145 (yTAF145) temperature-sensitive mutants in which transcription of a specific subset of genes was impaired at restrictive temperatures. The set of genes affected in these mutants overlapped with but was not identical to the set of genes affected by a previously reported yTAF145 mutant (W.-C. Shen and M. R. Green, Cell 90:615-624, 1997). To identify sequences which rendered transcription yTAF145 dependent, we conducted deletion analysis of the TUB2 promoter using a novel mini-CLN2 hybrid gene reporter system. The results showed that the yTAF145 mutations we isolated impaired core promoter recognition but did not affect activation by any of the transcriptional activators we tested. These observations are consistent with the reported yTAF145 dependence of the CLN2 core promoter in the mutant isolated by Shen and Green, although the CLN2 core promoter functioned normally in the mutants we report here. These results suggest that different promoters require different yTAF145 functions for efficient transcription. Interestingly, insertion of a canonical TATA element into the TATA-less TUB2 promoter rescued impaired transcription in the yTAF145 mutants we studied. It therefore appears that strong binding of TBP to the core promoter can alleviate the requirement for at least one yTAF145 function.

* Corresponding author. Mailing address: Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Phone: 81-743-72-5531. Fax: 81-743-72-5539. E-mail: kokubo{at}bs.aist-nara.ac.jp.

dagger Present address: Department of Biochemistry and Molecular Genetics, Health Sciences Center, University of Virginia, Charlottesville, VA 22908.

Molecular and Cellular Biology, April 2000, p. 2385-2399, Vol. 20, No. 7
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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