Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II:  Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

Abstract

CTD kinase I (CTDK-I) of Saccharomyces cerevisiae is required for normal phosphorylation of the C-terminal repeat domain (CTD) on elongating RNA polymerase II. To elucidate cellular roles played by this kinase and the hyperphosphorylated CTD (phosphoCTD) it generates, we systematically searched yeast extracts for proteins that bound to the phosphoCTD made by CTDK-I in vitro. Initially, using a combination of far-western blotting and phosphoCTD affinity chromatography, we discovered a set of novel phosphoCTD-associating proteins (PCAPs) implicated in a variety of nuclear functions. We identified the phosphoCTD-interacting domains of a number of these PCAPs, and in several test cases (namely, Set2, Ssd1, and Hrr25) adduced evidence that phosphoCTD binding is functionally important in vivo. Employing surface plasmon resonance (BIACORE) analysis, we found that recombinant versions of these and other PCAPs bind preferentially to CTD repeat peptides carrying SerPO 4 residues at positions 2 and 5 of each seven amino acid repeat, consistent with the positional specificity of CTDK-I in vitro [Jones, J. C., et al. (2004) J. Biol. Chem. 279, 24957-24964]. Subsequently, we used a synthetic CTD peptide with three doubly phosphorylated repeats (2,5P) as an affinity matrix, greatly expanding our search for PCAPs. This resulted in identification of approximately 100 PCAPs and associated proteins representing a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, protein synthesis and turnover, RNA degradation, snRNA modification, and snoRNP biogenesis). The varied nature of these PCAPs and associated proteins points to an unexpectedly diverse set of connections between Pol II elongation and other processes, conceptually expanding the role played by CTD phosphorylation in functional organization of the nucleus.The carboxyl-terminal repeat domain (CTD) 1 of the largest subunit of eukaryotic RNA polymerase II (Pol II) comprises tandem repeats of the consensus heptapeptide YSPTSPS. This highly conserved sequence, which is repeated 26 times in yeast and 52 times in mammals, is essential for viability. Phosphorylation of the CTD regulates the activities of the polymerase: only Pol II with an unphosphorylated CTD (Pol IIA) is thought to be able to assemble into preinitiation complexes at the promoter, whereas elongating polymerase has a hyperphosphorylated CTD (Pol IIO) (1,2). The serines at positions 2 and 5 within the heptad repeat represent major sites of phosphorylation during transcription. † Supported by National Institutes of Health Grant GM40505.© 2004 American Chemical Society * To whom correspondence should be addressed. . E-mail: arno@biochem.duke.edu. 1 Abbreviations: CTDK-I, CTD kinase I; Pol II, RNA polymerase II; CTD, C-terminal repeat domain; phosphoCTD, hyperphosphorylated CTD; PCTD, phosphoCTD; PCAP, phosphoCTD-associating protein; PCID, phosphoCTD-interacting domain; SGD, Saccharomyces Genome Database; SPR, surface plasmon resonance; RU, response units. NIH Publi...

Solution structure of the Set2–Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1

“…A GST-fusion protein carrying residues 1,884-2,061 of hSet2͞HYPB, containing a WW domain and a putative SRI domain, was expressed in and purified from bacterial cells (see Supporting Text, which is published as supporting information on the PNAS web site, for detailed procedures); far-Western blotting (performed as described in ref. 7) confirmed that this fusion protein binds the PCTD. Unexpectedly, thrombin cleavage released not only the intact hSet2 fragment but also a smaller piece that retained the ability to bind the PCTD (Fig.…”

“…Attendant with changes in patterns of CTD phosphorylation, the ensemble of CTD-bound proteins changes as RNA polymerase II progresses through the transcription cycle (5). Although knowledge of the number and types of PCTD-associating proteins (PCAPs) has expanded rapidly (7,8), information about the molecular nature of PCAP-PCTD interactions remains quite limited; detailed binding properties and͞or 3D structures are known for only a few PCTD-interacting domains (PCIDs) (9)(10)(11)(12)(13)(14).…”

Phosphorylation and functions of the RNA polymerase II CTD

“…A few of the other proteins identified in the biochemical search through the yeast proteome have already been shown to bind directly to the PCTD; these include Ess1, Prp40, Ssd1, and Hrr25 (Morris et al 1999;Morris and Greenleaf 2000;Phatnani et al 2004). Interestingly, the binding domains of three of these proteins bind best to Ser2,5P repeats, whereas the binding domain of Ssd1 binds equally well to Ser2,5P and Ser2P repeats ; the functional significance of these specificities have yet to be explored in vivo.…”

“…Finally, we point out that the order in which these interactions occur is not known; for instance, PCAPs that also bind RNA (e.g., Prp40, Cbf5) could bind the PCTD either before or after binding their cognate RNA. In addition, PCAP binding to the PCTD may be stabilized by interactions with other components of the megacomplex (e.g., Phatnani et al 2004, and its Supplementary Tables 1, 2).…”

Cross-talk Among RNA Polymerase II Kinases Modulates C-terminal Domain Phosphorylation

“…The pattern of phosphorylation on each heptad repeat and across the entire CTD is dynamic during transcription, resulting in transient and functionally distinct phospho-epitopes (3). These transient marks signal the spatio-temporal recruitment of over 100 different proteins with diverse roles (4).…”