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Molecular and Cellular Biology, August 2004, p. 7043-7058, Vol. 24, No. 16
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.16.7043-7058.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Célia Jeronimo,1,{dagger} Marie-France Langelier,1,{dagger} Mahel Zeghouf,2 Marilena Cojocaru,1 Dominique Bergeron,1 Dania Baali,1 Diane Forget,1 Sanie Mnaimneh,2 Armaity P. Davierwala,2 Jeff Pootoolal,2 Mark Chandy,3,4 Veronica Canadien,5 Bryan K. Beattie,5 Dawn P. Richards,5 Jerry L. Workman,3 Timothy R. Hughes,2 Jack Greenblatt,2 and Benoit Coulombe1*

Laboratory of Gene Transcription, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7,1 Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6,2 Affinium Pharmaceuticals, Toronto, Ontario M5J 1V6, Canada,5 Stowers Institute for Medical Research, Kansas City, Missouri 64110,3 Pennsylvania State University College of Medicine, Hershey, Pennsylvania 170334

Received 24 December 2003/ Returned for modification 9 February 2004/ Accepted 14 May 2004

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.

* Corresponding author. Mailing address: Laboratory of Gene Transcription, Institut de Recherches Cliniques de Montréal, 110 avenue des Pins Ouest, Montréal, Québec, Canada H2W 1R7. Phone: (514) 987-5662. Fax: (514) 987-5663. E-mail: coulomb{at}ircm.qc.ca.

{dagger} C.J. and M.-F.L. contributed equally to this work.

Molecular and Cellular Biology, August 2004, p. 7043-7058, Vol. 24, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.16.7043-7058.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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