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J. Biol. Chem., Vol. 276, Issue 49, 46408-46413, December 7, 2001

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Deletion of the RNA Polymerase Subunit RPB4 Acts as a Global, Not Stress-specific, Shut-off Switch for RNA Polymerase II Transcription at High Temperatures^*

Takenori Miyao, John D. Barnett, and Nancy A. Woychik^§

From the Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854-5635 and the  Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142

We used whole genome expression analysis to investigate the changes in the mRNA profile in cells lacking the Saccharomyces cerevisiae RNA polymerase II subunit RPB4 (RPB4). Our results indicated that an essentially complete shutdown of transcription occurs upon temperature shift of this conditionally lethal mutant; 98% of mRNA transcript levels decrease at least 2-fold, 96% at least 4-fold. This data was supported by in vivo experiments that revealed a rapid and greater than 5-fold decline in steady state poly(A) RNA levels after the temperature shift. Expression of several individual genes, measured by Northern analysis, was also consistent with the whole genome expression profile. Finally we demonstrated that the loss of RNA polymerase II activity causes secondary effects on RNA polymerase I, but not RNA polymerase III, transcription. The transcription phenotype of the RPB4 mutant closely mirrors that of the temperature-sensitive rpb1-1 mutant frequently implemented as a tool to inactivate the RNA polymerase II in vivo. Therefore, the RPB4 mutant can be used to easily design strains that enable the study of distinct post-transcriptional cellular processes in the absence of RNA polymerase II transcription.

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