Cloning and sequence determination of the<i>Schizosaccharomyces pombe rpb1</i>gene encoding the largest subunit of RNA polymerase II

Azuma, Yoshinao; Yamagishi, M; Ueshima, Rei; Ishihama, Akira

doi:10.1093/nar/19.3.461

Cited by 46 publications

(36 citation statements)

References 56 publications

(45 reference statements)

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“…The S. pombe pol II CTD consists of 29 heptad repeats preceded by two pseudorepeats (47). The S. pombe pol II CTD contains exclusively Ser 2 -Pro 3 or Ser 5 -Pro 6 dipeptides; there are no Thr-Pro dipeptides within the S. pombe CTD array.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

Pei

Shuman

2003

Journal of Biological Chemistry

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Schizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence 1 TPAWNSGSK 9 . We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1⌬ mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30°C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine.mRNA synthesis by RNA polymerase (pol) 1 II is regulated by phosphorylation of several of the protein components of the transcription apparatus (1). Phosphorylation of the carboxylterminal domain (CTD) of the largest subunit of pol II has been the focus of much attention because the CTD acts as an essential scaffold for the binding of macromolecular assemblies that regulate mRNA synthesis and cotranscriptional mRNA processing (2). The pol II CTD is composed of a heptapeptide motif of consensus sequence YSPTSPS repeated in tandem. The mammalian pol II CTD has 52 heptad repeats, the fission yeast Schizosaccharomyces pombe CTD has 29 repeats, and the budding yeast Saccharomyces cerevisiae CTD has 26 copies. The pol II CTD undergoes a cycle of extensive phosphorylation and dephosphorylation at positions Ser 5 and Ser 2 , which is coordinated with the transcription cycle (3). Multiple CTD kinases are present in eukaryotic cells, e.g. budding yeast has four CTD kinases, two of which (Kin28 and Bur1) are essential (3). CTD kinases are heteromeric enzymes consisting of a cyclin subunit plus a cyclin-dependent kinase (Cdk) subun...

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Section: Discussionmentioning

confidence: 99%

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

Pei

Shuman

2003

Journal of Biological Chemistry

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“…S. pombe RNA polymerase II consists of 10 putative subunits, which have been identified as homologues of the putative subunits, RPB1, RPB2, RPB3, RPB5, RPB6, RPB7, RPB8, RPB10, RPB11 and RPB12, of S. cerevisiae RNA polymerase II by amino acid micro sequences of isolated proteins, immunoreactivity and nucleotide sequences of the cloned genes (Azuma et al 1991;Kawagishi et al 1993;Azuma et al 1993;Shpakovski 1994;Sakurai et al 1996;Sakurai et al in preparation). Thus, it appears that subunits 4 and 9 are dispensable for the catalytic activity of RNA polymerization by S. pombe RNA polymerase II.…”

Section: Discussionmentioning

confidence: 99%

“…The three large subunits, Rpb1, Rpb2 and Rpb3, of S. pombe RNA polymerase II share sequence homologies with the 0 , and subunits, respectively, of prokaryotic RNA polymerases (Azuma et al 1991; T Miyao et al 35 S-labelled Rpb3, Rpb5 or luciferase synthesized in vitro in a reticulocyte lysate was mixed with the protein-immobilized beads. The bound proteins were analysed by SDS-12.5% PAGE and the gels were visualized with a BAS-2000 image analyser (Fuji).…”

Section: Discussionmentioning

confidence: 99%

“…These polypeptides have been tentatively designated as Pol II subunits (Woychik & Young 1994). On the other hand, purified RNA polymerase II from S. pombe contains 10 polypeptides (Sakurai et al 1996) and up to the present time, we have cloned and sequenced the genes encoding three large core subunits, Rpb1, Rpb2 and Rpb3 (Azuma et al 1991;Kawagishi et al 1993;Azuma et al 1993) and five small subunits, Rpb5, Rpb7, Rpb8, Rpb10, and Rpb11 (this paper, H. Sakurai & A. Ishihama, in preparation). Shpakovski (1994) and Shpakovski et al (1995) isolated the genes encoding Rpb6 and Rpb12.…”

Section: Introductionmentioning

confidence: 99%

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**Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5**

et al. 1996

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“…The fission yeast Schizosaccharomyces pombe is an attractive model system for CTD structure-function analysis because the native heptad repeat array is relatively homogeneous compared with other taxa. The S. pombe CTD usually is cited as consisting of 29 heptad repeats (12). The four most proximal repeats deviate in size and/or sequence from the consensus heptad, and the informational content of these deviant repeats is uncertain.…”

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confidence: 99%

Punctuation and syntax of the RNA polymerase II CTD code in fission yeast

Schwer

Sánchez

Shuman

2012

Proc. Natl. Acad. Sci. U.S.A.

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The primary structure and phosphorylation pattern of the tandem Y 3 and the spacing between coding units is flexible; the coding unit must contain two Tyr1 residues and the spacing between consecutive tyrosines is important; Ser5-PO 4 -Pro6 comprises an essential two-letter code "word" that is read by the mRNA capping apparatus; and a threshold number of Ser5-PO 4 -Pro6 words are needed to comprise a readable "sentence" of CTD information. Bypassing the essentiality of the Ser5 and Pro6 letters by fusion of capping enzymes to the CTD helped reveal how CTD phosphorylation circuits are wired in vivo. We found that the Ser2-PO 4 mark is independent of Ser5, Pro6, Ser7, and Thr4, whereas the Ser5-PO 4 mark is independent of Ser2, Ser7, and Thr4. These results provide unique insights to the reading and writing of the CTD code.CTD receptors | mRNA capping enzymes | serine phosphorylation T he carboxyl-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (Pol II) consists of tandemly repeated heptapeptides of the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 . The CTD is dispensable for RNA polymerase activity per se but is essential for cell viability because it recruits proteins that regulate transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation (1, 2). The inherently plastic CTD structure is modulated by phosphorylation of the heptad serine, threonine, and tyrosine residues. Phosphates are added by CTD kinases that have varying positional specificities and act at different stages of the transcription cycle. Phosphates are removed by diverse classes of CTD phosphatase enzymes that also have varying positional specificities and temporal windows of action during the transcription cycle.The combinatorial complexity of the CTD serine-2,5,7, threonine-4, and tyrosine-1 phosphorylation array comprises up to 32 n distinct primary structures, where n is the number of heptad repeats; cis-trans isomerization at CTD residues Pro3 and Pro6 imparts another 4 n conformational complexity. Thus, the total number of potential CTD structures is 128 n . The instantaneous primary structure of the CTD provides informational cues about the state of the transcription machinery-a CTD code-that is "read" by CTD receptor proteins (3). Whereas one may gain important insights to CTD coding principles by probing biochemically and structurally how individual receptor proteins recognize the CTD, it is technically impossible with present crude methods (and perhaps conceptually unattainable à la the uncertainty principle) to know the instantaneous primary structure of the CTD of a transcribing Pol II complex and the ensemble of proteins that are simultaneously engaged on the CTD.These issues notwithstanding, the informational rules that govern the CTD code on a cellular and organismal level can be probed genetically by manipulating the composition and structure of the Rpb1 CTD (4-11). The fission yeast Schizosaccharomyces pombe is an attractive model system for CTD structure-functi...

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Cloning and sequence determination of theSchizosaccharomyces pombe rpb1gene encoding the largest subunit of RNA polymerase II

Cited by 46 publications

References 56 publications

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

**Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5**

Punctuation and syntax of the RNA polymerase II CTD code in fission yeast

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