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Mol Cell Biol, April 1998, p. 1935-1945, Vol. 18, No. 4
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Analysis of the Interaction of the Novel RNA Polymerase II (pol II) Subunit hsRPB4 with Its Partner hsRPB7 and with pol II

Vladimir Khazak,^1, Joanne Estojak,¹ Helen Cho,² Jenifer Majors,¹ Gonosuke Sonoda,³ Joseph R. Testa,³ and Erica A. Golemis^1,*

Divisions of Basic Sciences¹ and Medical Sciences,³ Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and Department of Biochemistry, Howard Hughes Medical Institute, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854²

Received 25 August 1997/Returned for modification 7 October 1997/Accepted 26 January 1998

Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4 phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.

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^* Corresponding author. Mailing address: Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Phone: (215) 728-2860. Fax: (215) 728-3616. E-mail: EA_Golemis{at}fccc.edu.

Present address: Small Molecule Therapeutics, Monmouth Junction, N.J.

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