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Molecular and Cellular Biology, February 2000, p. 1263-1270, Vol. 20, No. 4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The Rpb6 Subunit of Fission Yeast RNA Polymerase II Is a Contact Target of the Transcription Elongation Factor TFIIS

Akira Ishiguro,1,2,3 Yasuhisa Nogi,2 Koji Hisatake,2 Masami Muramatsu,2 and Akira Ishihama3,*

School of Life Science, Graduate University for Advanced Studies,1 and Department of Molecular Genetics, National Institute of Genetics,3 Mishima, Shizuoka 411-8540, and Department of Biochemistry, Saitama Medical School, Moroyama, Iruma-Gun, Saitama 350-0095,2 Japan

Received 5 August 1999/Returned for modification 28 September 1999/Accepted 16 November 1999

The Rpb6 subunit of RNA polymerase II is one of the five subunits common to three forms of eukaryotic RNA polymerase. Deletion and truncation analyses of the rpb6 gene in the fission yeast Schizosaccharomyces pombe indicated that Rpb6, consisting of 142 amino acid residues, is an essential protein for cell viability, and the essential region is located in the C-terminal half between residues 61 and 139. After random mutagenesis, a total of 14 temperature-sensitive mutants were isolated, each carrying a single (or double in three cases and triple in one) mutation. Four mutants each carrying a single mutation in the essential region were sensitive to 6-azauracil (6AU), which inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AU sensitivity and temperature-sensitive phenotypes of these rpb6 mutants were suppressed by overexpression of TFIIS, a transcription elongation factor. In agreement with the genetic studies, the mutant RNA polymerases containing the mutant Rpb6 subunits showed reduced affinity for TFIIS, as measured by a pull-down assay of TFIIS-RNA polymerase II complexes using a fusion form of TFIIS with glutathione S-transferase. Moreover, the direct interaction between TFIIS and RNA polymerase II was competed by the addition of Rpb6. Taken together, the results lead us to propose that Rpb6 plays a role in the interaction between RNA polymerase II and the transcription elongation factor TFIIS.

* Corresponding author. Mailing address: National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411-8540, Japan. Phone: 81-559-81-6741. Fax: 81-559-81-6746. E-mail: aishiham{at}lab.nig.ac.jp.

Molecular and Cellular Biology, February 2000, p. 1263-1270, Vol. 20, No. 4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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