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Article

The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

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Pages 3259-3273 | Received 07 Mar 2013, Accepted 07 Jun 2013, Published online: 20 Mar 2023
 

Abstract

Transcription elongation factors associate with RNA polymerase II and aid its translocation through chromatin. One such factor is the conserved Paf1 complex (Paf1C), which regulates gene expression through several mechanisms, including the stimulation of cotranscriptional histone modifications. Previous studies revealed a prominent role for the Rtf1 subunit in tethering Paf1C to the RNA polymerase II elongation machinery. Here, we investigated the mechanism by which Rtf1 couples Paf1C to active chromatin. We show that a highly conserved domain of Rtf1 is necessary and sufficient for mediating a physical interaction between Rtf1 and the essential transcription elongation factor Spt5. Mutations that alter this Rtf1 domain or delete the Spt5 C-terminal repeat domain (CTR) disrupt the interaction between Rtf1 and Spt5 and release Paf1C from chromatin. When expressed in cells as the only source of Rtf1, the Spt5-interacting domain of Rtf1 can associate independently with active genes in a pattern similar to that of full-length Rtf1 and in a manner dependent on the Spt5 CTR. In vitro experiments indicate that the interaction between the Rtf1 Spt5-interacting domain and the Spt5 CTR is direct. Collectively, our results provide molecular insight into a key attachment point between Paf1C and the RNA polymerase II elongation machinery.

ACKNOWLEDGMENTS

We are grateful to Brett Tomson for her helpful comments on the manuscript and to Andrew VanDemark, Adam Wier, Christopher Amrich, Aubrey Lowen, Andrea Berman, Christopher Guerriero, Jeffrey Brodsky, and Margaret Shirra for technical assistance and advice. We thank Grant Hartzog for providing antisera against Spt5, Spt4-Spt5 expression plasmids, and yeast strains, Tim Formosa for Spt16 antibody, and Greg Prelich and Alan Hinnebusch for yeast strains. We are also grateful to Joe Martens and members of his laboratory for helpful suggestions throughout the course of this work and for sharing equipment and reagents.

This work was supported by grant NIH R01 GM52593 to K.M.A. and grant NIH/NCRR R21 RR025787 to R.G.G.

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