RPAP1, a Novel Human RNA Polymerase II-Associated Protein Affinity Purified with Recombinant Wild-Type and Mutated Polymerase Subunits

Abstract

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

We are grateful to the members of our laboratory for helpful discussions. We thank Diane Bourque for artwork and Julie Edwards for critical reading of the manuscript. We also thank Takahiro Nagase from the Kazusa DNA Research Institute for kindly providing the cDNA encoding RPAP1.

This work was supported by grants from the Canadian Institutes for Health Research (to B.C.), Genome Canada (to B.C., J.G., and T.R.H.), Genome Québec (to B.C.), the Ontario Genomics Institute (to J.G. and T.R.H.), and the Natural Sciences and Engineering Research Council of Canada (to T.R.H.). C.J. holds a studentship from the Canadian Institutes for Health Research and M.F.L. is supported by the Natural Sciences and Engineering Research Council of Canada and the Fonds Québécois de la Recherche sur la Nature et les Technologies. A.P.D. is supported by a C. H. Best Postdoctoral Fellowship. B.C. is a senior scholar from the Fonds de la Recherche en Santé du Québec.