Loss of the Rpb4/Rpb7 Subcomplex in a Mutant Form of the Rpb6 Subunit Shared by RNA Polymerases I, II, and III

Abstract

We have identified a conditional mutation in the shared Rpb6 subunit, assembled in RNA polymerases I, II, and III, that illuminated a new role that is independent of its assembly function. RNA polymerase II and III activities were significantly reduced in mutant cells before and after the shift to nonpermissive temperature. In contrast, RNA polymerase I was marginally affected. Although the Rpb6 mutant strain contained two mutations (P75S and Q100R), the majority of growth and transcription defects originated from substitution of an amino acid nearly identical in all eukaryotic counterparts as well as bacterial ω subunits (Q100R). Purification of mutant RNA polymerase II revealed that two subunits, Rpb4 and Rpb7, are selectively lost in mutant cells. Rpb4 and Rpb7 are present at substoichiometric levels, form a dissociable subcomplex, are required for RNA polymerase II activity at high temperatures, and have been implicated in the regulation of enzyme activity. Interaction experiments support a direct association between the Rpb6 and Rpb4 subunits, indicating that Rpb6 is one point of contact between the Rpb4/Rpb7 subcomplex and RNA polymerase II. The association of Rpb4/Rpb7 with Rpb6 suggests that analogous subunits of each RNA polymerase impart class-specific functions through a conserved core subunit.

ACKNOWLEDGMENTS

We thank Richard Ebright for numerous conversations and insights and for contributing panels A and B of Fig. 10; Michael Hampsey and Yuh-Hwa Wang for scientific discussions; and Bo-Shiun Chen for technical advice. We are grateful to Steve Hahn for kindly providing the Rpb9-TAP-tagged yeast strain. Gal4-VP16 was provided by the laboratory of Danny Reinberg. Finally, we thank Angela Then for sequencing the Rpb6 mutants and Keith McKune for technical assistance.

This work was funded by grant GM 55736 from the National Institutes of Health to N.A.W. M.H.P. was supported by a training grant, Virus-Host Interactions in Eukaryotic Cells from NIH-NIAID 2 T32 AI07403-10, awarded to Sidney Pestka.