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J. Biol. Chem., Vol. 278, Issue 51, 51566-51576, December 19, 2003

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The Conserved and Non-conserved Regions of Rpb4 Are Involved in Multiple Phenotypes in Saccharomyces cerevisiae^*

Vinaya Sampath, Nambudiry Rekha¶, N. Srinivasan||, and Parag Sadhale**

From the Department of Microbiology & Cell Biology and Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India

Rpb4, the fourth largest subunit of RNA polymerase II in Saccharomyces cerevisiae, is required for many phenotypes, including growth at high and low temperatures, sporulation, pseudohyphal growth, activated transcription of a subset of genes, and efficient carbon and energy metabolism. We have used deletion analysis to delineate the domains of the protein involved in these multiple phenotypes. The scRpb4 protein is conserved at the N and C termini but possesses certain non-conserved regions in the central portion. Our deletion analysis and molecular modeling results show that the N- and C-terminal conserved regions of Rpb4 are involved in interaction with Rpb7, the Rpb4 interacting partner in the RNA polymerase II. We further show that the conserved N terminus is required for efficient activated transcription from the INO1 promoter but not the GAL10- or the HSE-containing promoters. The N terminus is not required for any of the stress responses tested: growth at high temperatures, sporulation, and pseudohyphal growth. The conserved C-terminal 23 amino acids are not required for the role of Rpb4 in the pseudohyphal growth phenotype but might play a role in other stress responses and activated transcription. From the deletion analysis of the non-conserved regions, we report that they influence phenotypes involving both the N and C termini (interaction with Rpb7 and transcription from the INO1 promoter) but not any of the stress-responsive phenotypes tested suggesting that they might be involved in maintaining the two conserved domains in an appropriate conformation for interaction with Rpb7 and other proteins. Taken together, our results allow us to assign phenotype-specific roles for the different conserved and non-conserved regions of Rpb4.

Received for publication, June 4, 2003 , and in revised form, August 11, 2003.

^* This work has been supported in part by grants from the Council of Scientific and Industrial Research, the University Grants Commission, the Department of Science and Technology, and the Indian Council of Medical Research (to P. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a fellowship from Council of Scientific and Industrial Research, Government of India.

^|| Supported by the award of Senior Fellowship in Biomedical Sciences by the Wellcome Trust, UK.

^** To whom correspondence should be addressed. Tel.: 91-80-293-2292; Fax: 91-80-360-2697; E-mail: pps{at}mcbl.iisc.ernet.in.

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