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• W2019990809 abstract "The carboxyl-terminal domain (CTD) of elongating RNA polymerase II serves as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing. The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA and the one for which binding of the catalytic components is most clearly dependent on CTD phosphorylation. The present study highlights a distinctive strategy of cap targeting in fission yeast whereby the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the capping apparatus do not interact physically with each other (as they do in budding yeast and metazoans), but instead bind independently to the phosphorylated CTD. In vivointeractions of Pct1 and Pce1 with the CTD in a two-hybrid assay require 12 and 14 tandem repeats of the CTD heptapeptide, respectively. Pct1 and Pce1 bind in vitro to synthetic CTD peptides containing phosphoserine uniquely at position 5 or doubly at positions 2 and 5 of each of four tandem YSPTSPS repeats, but they bind weakly (Pce1) or not at all (Pct1) to a peptide containing phosphoserine at position 2. These results illustrate how remodeling of the CTD phosphorylation array might influence the recruitment and dissociation of the capping enzymes during elongation. But how does the CTD structure itself dictate interactions with the RNA processing enzymes independent of the phosphorylation state? Using CTD-Ser5 phosphopeptides containing alanine substitutions at other positions of the heptad, we define essential roles for Tyr-1 and Pro-3 (but not Thr-4 or Pro-6) in the binding ofSchizosaccharomyces pombeguanylyltransferase. Tyr-1 is also essential for binding and allosteric activation of mammalian guanylyltransferase by CTD Ser5-PO4, whereas alanine mutations of Pro-3 and Pro-6 reduce the affinity for the allosteric CTD-binding site. These are the first structure-activity relationships deduced for an effector function of the phosphorylated CTD. The carboxyl-terminal domain (CTD) of elongating RNA polymerase II serves as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing. The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA and the one for which binding of the catalytic components is most clearly dependent on CTD phosphorylation. The present study highlights a distinctive strategy of cap targeting in fission yeast whereby the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the capping apparatus do not interact physically with each other (as they do in budding yeast and metazoans), but instead bind independently to the phosphorylated CTD. In vivointeractions of Pct1 and Pce1 with the CTD in a two-hybrid assay require 12 and 14 tandem repeats of the CTD heptapeptide, respectively. Pct1 and Pce1 bind in vitro to synthetic CTD peptides containing phosphoserine uniquely at position 5 or doubly at positions 2 and 5 of each of four tandem YSPTSPS repeats, but they bind weakly (Pce1) or not at all (Pct1) to a peptide containing phosphoserine at position 2. These results illustrate how remodeling of the CTD phosphorylation array might influence the recruitment and dissociation of the capping enzymes during elongation. But how does the CTD structure itself dictate interactions with the RNA processing enzymes independent of the phosphorylation state? Using CTD-Ser5 phosphopeptides containing alanine substitutions at other positions of the heptad, we define essential roles for Tyr-1 and Pro-3 (but not Thr-4 or Pro-6) in the binding ofSchizosaccharomyces pombeguanylyltransferase. Tyr-1 is also essential for binding and allosteric activation of mammalian guanylyltransferase by CTD Ser5-PO4, whereas alanine mutations of Pro-3 and Pro-6 reduce the affinity for the allosteric CTD-binding site. These are the first structure-activity relationships deduced for an effector function of the phosphorylated CTD. polymerase II carboxyl-terminal domain binding domain polymerase chain reaction polyacrylamide gel electrophoresis mRNA capping occurs co-transcriptionally by a series of three enzymatic reactions in which the 5′-triphosphate terminus of the pre-mRNA is cleaved to a diphosphate by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and methylated at the N-7 position of guanine by RNA (guanine-7) methyltransferase (1Shuman S. Prog. Nucleic Acids Res. Mol. Biol. 2000; 66: 1-40Crossref Google Scholar). Specific targeting of cap formation to transcripts made by RNA polymerase II (pol II)1 is achieved, at least in part, through physical interactions of one or more components of the capping apparatus with the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of pol II (2McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 3Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 4Cho E. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (368) Google Scholar, 5Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar). The CTD, which is unique to pol II, is composed of a tandemly repeated heptad motif (consensus sequence = YSPTSPS). The mammalian pol II large subunit has 52 heptad repeats, the fission yeast Schizosaccharomyces pombe subunit has 29 repeats, and the budding yeastSaccharomyces cerevisiae subunit has 27 copies (6Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). The CTD undergoes a cycle of extensive phosphorylation and dephosphorylation of Ser-5 and Ser-2, which is coordinated with the transcription cycle. In the budding yeast S. cerevisiae, the guanylyltransferase (Ceg1) and methyltransferase (Abd1) components of the capping apparatus bind independently in vitro and in vivo to the phosphorylated CTD, but not to unphosphorylated CTD (2McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 7Schroeder S. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (297) Google Scholar). TheS. cerevisiae RNA triphosphatase Cet1 does not bind to the CTD by itself (8Cho E. Rodriguez C.R. Takagi T. Buratowski S. Genes Dev. 1998; 12: 3482-3487Crossref PubMed Scopus (85) Google Scholar), but it does bind to Ceg1 (9Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar, 10Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 11Ho C.K. Lehman K. Shuman S. Nucleic Acids Res. 1999; 27: 4671-4678Crossref PubMed Scopus (31) Google Scholar) and may thus be escorted to the transcription complex together with Ceg1. The mammalian capping enzyme Mce1 is a bifunctional polypeptide composed of an NH2-terminal RNA triphosphatase domain linked to a COOH-terminal guanylyltransferase domain. Mce1 binds to the pol II CTD and this interaction also requires CTD phosphorylation (2McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 5Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar). The guanylyltransferase domain Mce1(211–597) per se binds to CTD-PO4, but the NH2-terminal triphosphatase domain Mce1(1–210) does not (3Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 12Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Apparently, the covalent connection between the mammalian guanylyltransferase and triphosphatase domains allows the guanylyltransferase to deliver the triphosphatase to the transcription elongation complex. The mammalian guanylyltransferase can even deliver heterologous viral or fungal RNA triphosphatasesin vivo when they are fused to create chimeric capping enzymes (13Ho C.K. Martins A. Shuman S. J. Virol. 2000; 74: 5486-5494Crossref PubMed Scopus (26) Google Scholar). Recent studies indicate that the CTD and CTD phosphorylation play more than mere architectural roles in coordinating mRNA synthesis and processing (reviewed in Ref. 14Hirose Y. Manley J.L. Genes Dev. 2000; 14: 1415-1429PubMed Google Scholar). For example, the phosphorylated CTD directly stimulates the catalytic activity of the mammalian RNA guanylyltransferase (12Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 15Wen Y. Shatkin A.J. Genes Dev. 1999; 13: 1774-1779Crossref PubMed Scopus (163) Google Scholar). The remarkable feature of the activation of mammalian guanylyltransferase by the CTD is the requirement for phosphoserine at position 5. The guanylyltransferase also binds to CTD phosphorylated on Ser-2, but this interaction has no effect on enzyme activity (12Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Deletion analyses of the pol II large subunit have shown that the growth of fungal and metazoan cells is contingent on a minimal CTD length. Mammalian cells cannot grow with 25 or fewer CTD heptad repeats, but are fully viable with 36 repeats (16Bartolomei M.S. Halden N.F. Cullen C.R. Corden J.L. Mol. Cell. Biol. 1988; 8: 330-339Crossref PubMed Scopus (146) Google Scholar). West and Corden (17West M.L. Corden J.L. Genetics. 1995; 140: 1223-1233Crossref PubMed Google Scholar) found that truncating the S. cerevisiae CTD to 7 heptad repeats was lethal, whereas deletions leaving 8 or 9 heptads elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects, and normal cell growth required at least 10 integral copies of the heptad. A slightly higher threshold for yeast CTD length was reported by Nonet et al. (18Nonet M. Sweetser D. Young R.A. Cell. 1987; 50: 909-915Abstract Full Text PDF PubMed Scopus (200) Google Scholar), whereby less than 10 heptad repeat was lethal, 10 to 12 repeats resulted in conditional phenotypes, and 13 or more repeats sustained normal growth. Mutations that eliminate the hydroxyl moieties (potential phosphate acceptors) on Tyr-1, Ser-2, or Ser-5 of the heptad sequence are lethal to S. cerevisiae (17West M.L. Corden J.L. Genetics. 1995; 140: 1223-1233Crossref PubMed Google Scholar), but it is not clear if the loss of CTD function in vivo is a direct consequence of a perturbation of CTD structure (e.g. intra- or intermolecular loss of hydrogen bonding interactions) or a secondary effect on CTD phosphorylation state. There is relatively little known about the fine structure of the CTD and how such parameters as CTD length, amino acid sequence, and phosphorylation arrays influence various CTD-PO4 effector functions. The interaction of the capping apparatus with the phosphorylated CTD provides an attractive model system to address these issues. Here we present an analysis of the interactions of the capping enzymes of the fission yeast S. pombe with the CTD of the largest subunit of S. pombe pol II that highlights a new strategy for independent recruitment of separately encoded triphosphatase (Pct1) and guanylyltransferase (Pce1) components to the pol II elongation complex. Analysis of the interaction of S. pombe and mammalian capping enzymes with mutated synthetic CTD phosphopeptides illuminates for the first time the contributions of conserved CTD side chains to CTD function independent of any effects on CTD phosphorylation. This approach should be applicable to the study of CTD-PO4 effector roles in other co-transcriptional RNA transactions such as pre-mRNA splicing, polyadenylation, and transcription termination. The screen was performed using the Matchmaker system from CLONTECH with protocols specified by the vendor. The full-length PCT1 andPCE1 genes were inserted into pAS2-1 (2µ TRP1) so as to fuse them in-frame with the GAL4 DNA-binding domain (BD). The BD-PCT1 and BD-PCE1 plasmids were transformed into S. cerevisiae strain Y190. Trp+isolates were selected and grown in 1-liter cultures SD(−Trp) medium. The cells were harvested and transformed by the LiCl method with anS. pombe cDNA library in a GAL4 activation domain (AD) fusion plasmid (2µ LEU2) (19Cooper J.P. Nimmo E.R. Allshire R.C. Cech T.R. Nature. 1997; 385: 744-747Crossref PubMed Scopus (434) Google Scholar). The transformant pool was selected directly for HIS3 reporter expression by plating on SD(-Trp, -Leu, -His) agar containing 25 mm 3-amino-1,2,4-triazole. The total number of transformants screened was estimated by plating an aliquot of the pool on SD(-Trp, -Leu) agar. The His+ colonies were selected after incubation for 3 to 7 days at 30 °C, then patched and restreaked on SD(-Trp, -Leu, -His, 3-amino-1,2,4-triazole) agar. Single colonies that retested for His+ were patched and tested forlacZ reporter expression using the β-galactosidase colony-lift filter assay. DNA recovered from the strains that tested positive for both HIS3 and lacZ expression was used as the template for PCR amplification of the S. pombeDNA insert with flanking primers specific for the AD-fusion plasmid. The PCR products were gel-purified and then sequenced. The AD plasmid clones were recovered after transformation into Escherichia coli DH5α. Gene fragments encoding serially truncated versions of S. pombe Rpb1 were generated by PCR amplification using antisense primers that introduced stop codons in lieu of the codons for amino acids 1687, 1669, 1648, 1634, 1592, or 1585 and a XhoI site immediately 3′ of the stop codon. The sense primer introduced a BamHI site at the codon for amino acid 1516. The PCR products were digested with BamHI andXhoI and then inserted into the two-hybrid AD fusion vector pGAD-GH. Sequencing of the inserts in the resulting series of AD-Rbp1 plasmids verified that the truncated RPB1 genes were fused in-frame to AD and that no unwanted coding changes had been introduced during amplification and cloning. S. pombe RNA triphosphatase Pct1 and mouse guanylyltransferase Mce1(211–597) were produced in E. coli as N-terminal His-tagged fusions and purified from soluble bacterial lysates by nickel-agarose chromatography as described previously (3Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 20Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar). The Mce1(211–597)-[32P]GMP intermediate was prepared by reaction of 20 µg of Mce1(211–597) with 5 µm [α-32P]GTP and 2.5 mmMgCl2 for 30 min at 30 °C. The reaction mixture was adjusted to 25 mm EDTA and 10% glycerol and the native enzyme-GMP intermediate resolved from free [32P]GTP by gel filtration through a 1-ml column of Sephadex G-50. S. pombe RNA guanylyltransferase Pce1 was produced inE. coli as a His-tagged fusion as follows. The pET-PCE1 plasmid (21Shuman S. Liu Y. Schwer B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12046-12050Crossref PubMed Scopus (96) Google Scholar) was modified by insertion of a DNA fragment encoding an NH2-terminal 21-amino acid peptide (MGHHHHHHHHHHSSGHIEGRP) in-frame with the open reading frame encoding the 402-amino acid Pce1 polypeptide. The pETHis-PCE1 plasmid was transformed into E. coli BL21(DE3). A 250-ml culture was grown at 37 °C in LB medium containing 0.1 mg/ml ampicillin until theA 600 reached 0.5. The culture was adjusted to 0.4 mm isopropyl-1-thio-β-d-galactopyranoside and 2% ethanol and incubation was continued for 20 h at 17 °C. Cells were harvested by centrifugation and stored at −80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 25 ml of buffer A (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 10% glycerol). Phenylmethylsulfonyl fluoride and lysozyme were added to final concentrations of 300 µmand 100 µg/ml, respectively. After incubation on ice for 30 min, Triton X-100 was added to a final concentration of 0.1% and the lysate was sonicated to reduce viscosity. Insoluble material was removed by centrifugation for 45 min at 18,000 rpm in a Sorvall SS34 rotor. The soluble extract was mixed for 30 min with 1 ml of Ni2+-NTA-agarose (Qiagen) that had been equilibrated with buffer A containing 0.1% Triton X-100 and 25 mm imidazole. The slurry was poured into a column and the resin was washed with 10 ml of buffer A containing 0.1% Triton X-100 and 50 mmimidazole. Pce1 was then step-eluted with 250 mm imidazole in buffer A. The enzyme preparation (7 mg of protein) was dialyzed against buffer containing 50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 1 mm dithiothreitol, 5% glycerol, 0.03% Triton X-100, and stored at −80 °C. The protein concentration was determined using the Bio-Rad dye-binding method with bovine serum albumin as the standard. NH2-terminal biotinylated CTD phosphopeptides composed of 4 tandem YSPTSPS repeats containing phosphoserine at position 2, position 5, or positions 2 plus 5 of each repeat were synthesized and purified as described previously (12Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 22Schwer B. Lehman K. Saha N. Shuman S. J. Biol. Chem. 2001; 276: 1857-1864Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Mutant Ser-5 phosphopeptides Y1A (ASPTSPS)4, P3A (YSATSPS)4, T4A (YSPASPS)4, and P6A (YSPSTAS)4 were synthesized and purified using the same methods. The molecular weights of the mutant phosphopeptides were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The measured masses were in agreement with the calculated theoretical masses within the limits of calibration of the instrument. The peptides were dissolved in 10 mm Tris-HCl (pH 8.0), 1 mm EDTA and stored at 4 °C. The biotinylated CTD peptides were absorbed to streptavidin-coated magnetic beads (Dynabeads M280 streptavidin; Dynal) in binding buffer (25 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol, 5% glycerol, 0.03% Triton X-100) containing 50 mm NaCl as described previously (12Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The amount of biotinylated CTD peptide was sufficient to saturate the bead-bound streptavidin. After peptide adsorption, the beads were washed three times with binding buffer to remove any unbound peptide. Affinity chromatography was performed by mixing 14 µg (280 pmol) of Pce1, 9 µg (240 pmol) of Pct1, or 4 µg (85 pmol) of Mce1(211–597) with 0.6 mg of CTD peptide beads (estimated to contain 390 pmol of peptide) in 50 µl of binding buffer with 50 mm NaCl. After incubation for 30 min on ice, the beads were concentrated by microcentrifugation for 15 s and then held in place with a magnet as the supernatant was withdrawn. The beads were resuspended in 1 ml of binding buffer and subjected to two cycles of concentration and washing. After the third wash, the beads were resuspended in 50 µl of binding buffer. Aliquots (20 µl) of the bead bound fraction were mixed with 4 µl of SDS sample buffer (200 mm Tris-HCl, pH 6.8, 8% SDS, 140 nmβ-mercaptoethanol, 40% glycerol), heated at 90 °C for 3–5 min, and then analyzed by SDS-PAGE. The triphosphatase and guanylyltransferase components of fungal capping systems have conserved catalytic domains (1Shuman S. Prog. Nucleic Acids Res. Mol. Biol. 2000; 66: 1-40Crossref Google Scholar), but the enzymes are not always functionally interchangeablein vivo. S. pombe and Candida albicansguanylyltransferases can function in S. cerevisiae with the endogenous Cet1 triphosphatase (21Shuman S. Liu Y. Schwer B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12046-12050Crossref PubMed Scopus (96) Google Scholar, 23Yamada-Okabe T. Shimmi O. Doi R. Mizumoto K. Arisawa M. Yamada-Okabe H. Microbiology. 1996; 142: 2515-2523Crossref PubMed Scopus (45) Google Scholar), but the ability of heterologous triphosphatases to function with the S. cerevisiae guanylyltransferase is variable and correlates with the presence or absence of a conserved guanylyltransferase-binding domain on the surface of the heterologous RNA triphosphatase (11Ho C.K. Lehman K. Shuman S. Nucleic Acids Res. 1999; 27: 4671-4678Crossref PubMed Scopus (31) Google Scholar, 24Lima C.D. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar).S. pombe triphosphatase Pct1 (a 303-amino acid polypeptide), which lacks the surface domain and does not function on its own inS. cerevisiae, can support cell growth when S. pombe guanylyltransferase Pce1 (a 402-amino acid polypeptide) is provided in trans (20Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar). Thus, the fission yeast capping components display a species-specific genetic interaction. Given that the triphosphatase and guanylyltransferase components of the capping apparatus are physically associated in budding yeast and metazoans (with the interaction being noncovalent in S. cerevisiae and covalent in metazoans), the simplest model to account for the genetic interaction would be that S. pombetriphosphatase Pct1 forms a complex with S. pombeguanylyltransferase Pce1 and that Pce1, which binds in vitroto CTD-PO4 (2McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar), in turn chaperones the triphosphatase to the transcription complex. To test this hypothesis, we asked whether a mixture of recombinant Pct1 (a homodimer) and recombinant Pce1 (a monomeric protein) resulted in the formation of a stable heteromeric complex that could be isolated by velocity sedimentation using the methods that readily detect formation of a complex between S. cerevisiae Cet1 and Ceg1 (10Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We were unable to detect any complex formation between the S. pombe Pct1 and Pce1 (not shown). Next, we conducted a two-hybrid screen of a S. pombecDNA library linked to the Gal4 activation domain (AD) using as “bait” either Pce1 or Pct1 fused to the Gal4 DNA-BD. Although the screens yielded multiple interacting clones in each case (see below), we did not isolate Pce1 or a fragment thereof using Pct1 as bait. Nor did we isolate Pct1 or a fragment thereof using Pce1 as bait. To exclude the possibility of a false negative caused by absence of the Pce1 or Pct1 genes from the S. pombe plasmid library, we performed a directed two-hybrid interaction assay using an explicitly engineered pair of BD-Pce1 and AD-Pct1 vectors. Here again, we saw no evidence of interaction between the S. pombe triphosphatase and guanylyltransferase (not shown). Note that the S. cerevisiae and C. albicans triphosphatase and guanylyltransferase components display two-hybrid interactions with one another (25Yamada-Okabe T. Mio T. Matsui M. Kashima Y. Arisawa M. Yamada-Okabe H. FEBS Lett. 1998; 435: 49-54Crossref PubMed Scopus (30) Google Scholar). A two-hybrid screen of ∼100,000 transformants for triphosphatase-interacting proteins using BD-Pct1 as bait yielded 16 His+ isolates, of which 8 contained plasmids encoding AD fused in-frame to a COOH-terminal fragment of Rpb1, the largest subunit of S. pombe pol II (26Azuma Y. Yamagashi M. Ueshima R. Ishihama A. Nucleic Acids Res. 1991; 19: 461-468Crossref PubMed Scopus (47) Google Scholar). Three different AD-Rpb1 fusion clones were isolated: AD-Rpb1(1305–1752) was recovered twice; AD-Rpb1(1324–1752) was recovered 5 times; and AD-Rpb1(1478–1752) was isolated once (Fig. 1). Control experiments showed that the His+ andlacZ + phenotypes required co-transformation with BD-Pct1 and AD-Rpb1(1305–1752) plasmids and that neither fusion plasmid was able to drive expression of the HIS3 orlacZ reporter genes when co-transformed with the BD or AD vectors (Fig. 2).Figure 2Specificity of the Pct1-CTD two-hybrid interaction. The BD-Pct1 and AD-Rpb1(1305–1752) fusion plasmids and the BD and AD vectors without inserts were transformed pairwise into S. cerevisiae Y190. Panel A, single Trp+ Leu+ isolates containing the indicated plasmid pairs were selected and streaked on SD(-Trp, -Leu, -His, 3-amino-1,2,4-triazole) agar medium. The plate was photographed after incubation for 5 days at 30 °C. Panel B, Trp+Leu+ isolates containing the indicated plasmid pairs were patched on SD(-Trp, -Leu) agar medium. The cell patches were photographed after incubation for 1 day at 30 °C (Growth). The cell patches were then tested for β-galactosidase activity using the colony-lift filter assay. A photograph of the filter is shown (lacZ).View Large Image Figure ViewerDownload (PPT) A two-hybrid screen of ∼200,000 transformants for guanylyltransferase-interacting proteins using BD-Pce1 as bait yielded 11 His+ isolates. Sequencing of the AD plasmids from these strains showed that 8 of the isolates encoded AD fused in-frame to a COOH-terminal fragment of Rpb1 from residues 1324 to 1752 (Fig. 1). Control transformations confirmed that HIS3 andlacZ expression required co-transformation with the BD-Pce1 and AD-Rpb1(1324–1752) plasmids (not shown). The AD-Rpb1 fusions that interacted in vivo with Pce1 or Pct1 contained all 29 tandem copies of the heptad repeat plus a variable segment of the pol II subunit upstream of the start of the CTD at position 1551 (26Azuma Y. Yamagashi M. Ueshima R. Ishihama A. Nucleic Acids Res. 1991; 19: 461-468Crossref PubMed Scopus (47) Google Scholar). To gauge the role of the non-reiterated protein segment, we constructed an AD-Rpb1(1516–1752) fusion clone and tested it in a directed two-hybrid assay paired with BD-Pce1 and BD-Pct1. We found that the Rpb1 interaction with both capping enzymes persisted when the AD fusion contained little more than the CTD repeats per se. Thus, subsequent COOH-terminal truncations of the CTD were performed with the NH2-terminal position of Rpb1 fixed at 1516 (Fig. 1). The salient findings were that the CTD interaction with Pct1 persisted with undiminished strength (as gauged by colony size during selection for His+) with 19, 17, or 14 heptad repeats, with diminished strength after truncation to 12 heptad repeats, and was eliminated with 6 or fewer heptads (Fig. 1). The two-hybrid CTD interaction with Pce1 displayed a more stringent requirement for CTD length; the interaction was of diminished strength at between 19 and 14 heptad repeats and was abolished at 12 or fewer repeats (Fig. 1). Previous CTD-binding studies in vitroinvolving S. pombe guanylyltransferase employed an immobilized ligand composed of recombinant GST-CTD that was enzymatically phosphorylated in vitro using HeLa cell extract as the source of CTD kinase. The fusion protein contained 15 copies of the YSPTSPS heptad sequence and an average of 3 serine-phosphates per GST-CTD polypeptide (2McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar). We have since developed synthetic CTD phosphopeptide ligands, in which the numbers and positions of the phosphates are known and subject to manipulation, to delineate the requirements for the interaction of capping enzymes with the CTD (12Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Here we employed 28-mer CTD peptide ligands composed of four tandem repeats of the YSPTSPS heptad that contained phosphoserine at either position 2 or 5 of each heptad or phosphoserine atboth positions 2 and 5 of each heptad. An NH2-terminal biotin moiety was added during chemical synthesis so that the peptides could be linked to streptavidin-coated beads for affinity chromatography purposes. The CTD phosphopeptide-containing beads, or control beads containing an unphosphorylated 28-mer CTD peptide, were incubated with purified recombinant Pce1. The beads were recovered by centrifugation and washed three times with buffer. The bead-bound material was eluted with 1% SDS. The input guanylyltransferase protein (L) and the bead-bound fractions were then analyzed by SDS-PAGE. The key findings were that the S. pombe guanylyltransferase bound equally well to the CTD Ser5-PO4 peptide and the Ser2-PO4/Ser5-PO4 peptide. Slightly less than one-fifth of the input protein was retained on the beads in both cases. Pce1 bound less avidly to the CTD Ser2-PO4 peptide. The binding was specific for CTD-PO4, because Pce1 did not bind at all to the beads containing the unphosphorylated CTD peptide (Fig.3A). Potential effects of the CTD Ser2-PO4/Ser5-PO4 peptide on Pce1 activity were gauged by preincubating recombinant Pce1 with increasing amounts of the peptide (up to 120-fold molar excess of peptide over Pce1) and then assaying the mixtures for enzyme-GMP complex formation during a reaction in vitro with [α-32P]GTP. The phosphopeptide had no significant stimulatory or inhibitory effect on Pce1 activity (not shown). The novel finding was that the recombinant S. pombe RNA triphosphata" @default.
• W2019990809 created "2016-06-24" @default.
• W2019990809 creator A5014112082 @default.
• W2019990809 creator A5025520245 @default.
• W2019990809 creator A5026038727 @default.
• W2019990809 creator A5081913602 @default.
• W2019990809 creator A5088178390 @default.
• W2019990809 date "2001-07-01" @default.
• W2019990809 modified "2023-10-09" @default.
• W2019990809 title "The Length, Phosphorylation State, and Primary Structure of the RNA Polymerase II Carboxyl-terminal Domain Dictate Interactions with mRNA Capping Enzymes" @default.
• W2019990809 cites W1591148945 @default.
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