A Novel Collection of Accessory Factors Associated with Yeast RNA Polymerase II

Wade, Paul A.; Werel, W.; Fentzke, Richard C.; Thompson, Nancy E.; Leykam, Joseph F.; Burgess, Richard R.; Jaehning, Judith A.; Burton, Zachary F.

doi:10.1006/prep.1996.0077

Cited by 99 publications

(102 citation statements)

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“…These two proteins are components of the Paf1 complex which associates with transcriptionally active genes (110). This complex was originally postulated to bind to the body of Pol II, independent of the CTD (43). Therefore, CTD phosphorylation may not play a role in their recruitment; this is consistent with recent data indicating that Ctr9 and other Paf1 components can be cross-linked to genes independent of Ctk1 (4).…”

Section: Pcaps and Associated Proteins From P11 Fractions: Expanding supporting

confidence: 84%

“…This ionic strength [0.4 M (NH 4 ) 2 SO 4 ] has been shown to be sufficient to extract factors associated with elongating Pol II from yeast (42,43), and we have found that it is also more than sufficient to disengage virtually all mammalian PCAPs from RNA Pol II in a chromatin pellet from HeLa cells (39). The crude extract was centrifuged at 14000g (8000 rpm in a Sorvall SLC-6000 rotor) for 45 min at 4 °C to remove cell debris.…”

Section: (C) Methods 3: P11 Fractionationmentioning

confidence: 99%

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

2004

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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...

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Section: Pcaps and Associated Proteins From P11 Fractions: Expanding supporting

confidence: 84%

Section: (C) Methods 3: P11 Fractionationmentioning

confidence: 99%

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

2004

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“…2A-C). The latter fragment of parafibromin contains the region from residues 233 to 525 homologous to cdc73p, the yeast RNA polymerase II accessory factor component of the Paf1 complex (20,21). Because yeast cdc73p and the other yeast Paf1 complex components are nuclear proteins (22), and because human parafibromin (hCdc73) has been identified as a component of a human PAF1 complex (12)(13)(14), it was expected that GFP-HRPT2-C would exhibit nuclear localization like the full-length parafibromin fusion (GFP-HRPT2-A).…”

Section: Resultsmentioning

confidence: 99%

Nuclear Localization of the Parafibromin Tumor Suppressor Protein Implicated in the Hyperparathyroidism-Jaw Tumor Syndrome Enhances Its Proapoptotic Function

Czapiga

Nini

et al. 2007

Molecular Cancer Research

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Parafibromin is a tumor suppressor protein encoded by HRPT2, a gene recently implicated in the hereditary hyperparathyroidism-jaw tumor syndrome, parathyroid cancer, and a subset of kindreds with familial isolated hyperparathyroidism. Human parafibromin binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex. The mechanism by which loss of parafibromin function can lead to neoplastic transformation is poorly understood. Because the subcellular localization of parafibromin is likely to be critical for its function with the nuclear PAF1 complex, we sought to experimentally define the nuclear localization signal (NLS) of parafibromin and examine its potential role in parafibromin function. Using site-directed mutagenesis, we define a dominant bipartite NLS and a secondary NLS, both in the NH 2 -terminal region of parafibromin whose combined mutation nearly abolishes nuclear targeting. The NLS-mutant parafibromin is significantly impaired in its association with endogenous Paf1 and Leo1. We further report that overexpression of wild-type but not NLS-mutant parafibromin induces apoptosis in transfected cells. Inhibition of endogenous parafibromin expression by RNA interference inhibits the basal rate of apoptosis and apoptosis resulting from DNA damage induced by camptothecin, a topoisomerase I inhibitor. These experiments identify for the first time a proapoptotic activity of endogenous parafibromin likely to be important in its role as a tumor suppressor and show a functional role for the NLS of parafibromin in this activity. (Mol Cancer Res 2007;5(2):183 -93)

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“…This region alone can interact with RNAPII-containing complexes during affinity chromatography (Pan et al 1997), suggesting that domain I may facilitate interactions between TFIIS and other proteins found in one or more holoenzyme complexes. For example, TFIIS is physically associated with a Paf1p-containing RNAPII complex (Wade et al 1996) and PPR2 exhibits genetic interactions with PAF1 (Squazzo et al 2002). However, the requirement for TFIIS domain I in these physical and genetic interactions remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

Genetic Interactions Between TFIIF and TFIIS

et al. 2006

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The eukaryotic transcript elongation factor TFIIS is encoded by a nonessential gene, PPR2, in Saccharomyces cerevisiae. Disruptions of PPR2 are lethal in conjunction with a disruption in the nonessential gene TAF14/TFG3. While investigating which of the Taf14p-containing complexes may be responsible for the synthetic lethality between ppr2D and taf14D, we discovered genetic interactions between PPR2 and both TFG1 and TFG2 encoding the two larger subunits of the TFIIF complex that also contains Taf14p. Mutant alleles of tfg1 or tfg2 that render cells cold sensitive have improved growth at low temperature in the absence of TFIIS. Remarkably, the amino-terminal 130 amino acids of TFIIS, which are dispensable for the known in vitro and in vivo activities of TFIIS, are required to complement the lethality in taf14D ppr2D cells. Analyses of deletion and chimeric gene constructs of PPR2 implicate contributions by different regions of this N-terminal domain. No strong common phenotypes were identified for the ppr2D and taf14D strains, implying that the proteins are not functionally redundant. Instead, the absence of Taf14p in the cell appears to create a dependence on an undefined function of TFIIS mediated by its N-terminal region. This region of TFIIS is also at least in part responsible for the deleterious effect of TFIIS on tfg1 or tfg2 cold-sensitive cells. Together, these results suggest a physiologically relevant functional connection between TFIIS and TFIIF.

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A Novel Collection of Accessory Factors Associated with Yeast RNA Polymerase II

Cited by 99 publications

References 0 publications

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

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

Nuclear Localization of the Parafibromin Tumor Suppressor Protein Implicated in the Hyperparathyroidism-Jaw Tumor Syndrome Enhances Its Proapoptotic Function

Genetic Interactions Between TFIIF and TFIIS

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