Structure-Function Relationship of Yeast S-II in Terms of Stimulation of RNA Polymerase II, Arrest Relief, and Suppression of 6-Azauracil Sensitivity
1995; Elsevier BV; Volume: 270; Issue: 15 Linguagem: Inglês
10.1074/jbc.270.15.8991
ISSN1083-351X
AutoresToshiyuki Nakanishi, Makoto Shimoaraiso, Takeo Kubo, Shunji Natori,
Tópico(s)Synthesis and Biological Evaluation
ResumoThe yeast S-II null mutant is viable, but the mutation induces sensitivity to 6-azauracil. To examine whether the region needed for stimulation of RNA polymerase II and that for suppression of 6-azauracil sensitivity in the S-II molecule could be separated, we constructed various deletion mutants of S-II and expressed them in the null mutant using the GAL1 promoter to see if the mutant proteins suppressed 6-azauracil sensitivity. We also expressed these constructs in Escherichia coli, purified the mutant proteins to homogeneity, and examined if they stimulated RNA polymerase II. We found that a mutant protein lacking the first 147 amino acid residues suppressed 6-azauracil sensitivity but that removal of 2 additional residues completely abolished the suppression. A mutant protein lacking the first 141 residues had activity to stimulate RNA polymerase II, whereas removal of 10 additional residues completely abolished this activity. We also examined arrest-relief activity of these mutant proteins and found that there is a good correlation between RNA polymerase II-stimulating activity and arrest-relief activity. Therefore, at least the last 168 residues of S-II are sufficient for expressing these three activities. The yeast S-II null mutant is viable, but the mutation induces sensitivity to 6-azauracil. To examine whether the region needed for stimulation of RNA polymerase II and that for suppression of 6-azauracil sensitivity in the S-II molecule could be separated, we constructed various deletion mutants of S-II and expressed them in the null mutant using the GAL1 promoter to see if the mutant proteins suppressed 6-azauracil sensitivity. We also expressed these constructs in Escherichia coli, purified the mutant proteins to homogeneity, and examined if they stimulated RNA polymerase II. We found that a mutant protein lacking the first 147 amino acid residues suppressed 6-azauracil sensitivity but that removal of 2 additional residues completely abolished the suppression. A mutant protein lacking the first 141 residues had activity to stimulate RNA polymerase II, whereas removal of 10 additional residues completely abolished this activity. We also examined arrest-relief activity of these mutant proteins and found that there is a good correlation between RNA polymerase II-stimulating activity and arrest-relief activity. Therefore, at least the last 168 residues of S-II are sufficient for expressing these three activities. Transcription factor S-II, originally isolated from Ehrlich ascites tumor cells as a specific stimulator of RNA polymerase II, has been studied extensively (1Sekimizu K. Kobayashi N. Mizuno D. Natori S. Biochemistry. 1976; 15: 5064-5070Crossref PubMed Scopus (65) Google Scholar, 2Sekimizu K. Nakanishi Y. Mizuno D. Natori S. Biochemistry. 1979; 18: 1582-1588Crossref PubMed Scopus (56) Google Scholar, 3Sekimizu K. Yokoi H. Natori S. J. Biol. Chem. 1982; 257: 2719-2721Abstract Full Text PDF PubMed Google Scholar, 4Ueno K. Sekimizu K. Mizuno D. Natori S. Nature. 1979; 277: 145-146Crossref PubMed Scopus (33) Google Scholar, 5Natori S. Mol. Cell. Biochem. 1982; 46: 173-187Crossref PubMed Scopus (33) Google Scholar, 6Horikoshi M. Sekimizu K. Natori S. J. Biol. Chem. 1984; 259: 608-611Abstract Full Text PDF PubMed Google Scholar, 7Horikoshi M. Sekimizu K. Hirashima S. Mitsuhashi Y. Natori S. J. Biol. Chem. 1985; 260: 5739-5744Abstract Full Text PDF PubMed Google Scholar, 8Rappaport J. Reinberg D. Zandomein R. Weinmann R. J. Biol. Chem. 1987; 262: 5227-5232Abstract Full Text PDF PubMed Google Scholar, 9Reinberg D. Roeder R.G. J. Biol. Chem. 1987; 262: 3331-3337Abstract Full Text PDF PubMed Google Scholar, 10Hirashima S. Hirai H. Nakanishi Y. Natori S. J Biol. Chem. 1988; 263: 3858-3863Abstract Full Text PDF PubMed Google Scholar, 11Sluder A.E. Greenleaf A.L. Price D.H. J. Biol. Chem. 1989; 264: 8963-8969Abstract Full Text PDF PubMed Google Scholar, 12Bengal E. Flores O. Krauskopf A. Reinberg D. Aloni Y. Mol. Cell. Biol. 1991; 11: 1195-1206Crossref PubMed Scopus (116) Google Scholar, 13Kanai A. Kuzuhara T. Sekimizu K. Natori S. J. Biochem. 1991; 109: 674-677Crossref PubMed Scopus (25) Google Scholar, 14Wiest D.K. Wang D. Hawley D.K. J. Biol. Chem. 1992; 267: 7733-7744Abstract Full Text PDF PubMed Google Scholar, 15Xu Q. Nakanishi T. Sekimizu K. Natori S. J. Biol. Chem. 1994; 269: 3100-3103Abstract Full Text PDF PubMed Google Scholar). Results have shown that S-II is one of the transcription elongation factors that promote read-through by RNA polymerase II of pausing sites within genes in vitro (16Reines D. Chamberlin M.J. Kane C.M. J. Biol. Chem. 1989; 264: 10799-10809Abstract Full Text PDF PubMed Google Scholar, 17SivaRaman L. Reines D. Kane C.M. J. Biol. Chem. 1990; 265: 14554-14560Abstract Full Text PDF PubMed Google Scholar). S-II has been proposed to release pausing by inducing cleavage of the 3′-end of the nascent transcript in the ternary elongation complex (Refs. 18Izban M.G. Luse D.S. Genes & Dev. 1992; 6: 1342-1356Crossref PubMed Scopus (227) Google Scholar, 19Izban M.G. Luse D.S. J. Biol. Chem. 1993; 268: 12864-12873Abstract Full Text PDF PubMed Google Scholar, 20Reines D. J. Biol. Chem. 1992; 267: 3795-3800Abstract Full Text PDF PubMed Google Scholar, 21Reines D. Ghanouni P. Li Q.-q Mote Jr., J. J. Biol. Chem. 1992; 267: 15516-15522Abstract Full Text PDF PubMed Google Scholar, 22Reines D. Mote Jr., J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1917-1921Crossref PubMed Scopus (92) Google Scholar, 23Gu W. Powell W. Mote Jr., J. Reines D. J. Biol. Chem. 1993; 268: 25604-25616Abstract Full Text PDF PubMed Google Scholar-24Guo H. Price D.H. J. Biol. Chem. 1993; 268: 18762-18770Abstract Full Text PDF PubMed Google Scholar; for review, see Ref. 25Kassavatis G.A. Geiduschek E.P. Science. 1993; 259: 944-945Crossref PubMed Scopus (66) Google Scholar). Recently, we purified S-II from yeast (Saccharomyces cerevisiae) and determined its complete amino acid sequence by isolating its gene (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar). Significant sequence similarity was found between yeast S-II and other S-IIs so far sequenced (10Hirashima S. Hirai H. Nakanishi Y. Natori S. J Biol. Chem. 1988; 263: 3858-3863Abstract Full Text PDF PubMed Google Scholar, 27Marshall T.K. Guo H. Price D.H. Nucleic Acids Res. 1990; 18: 6293-6298Crossref PubMed Scopus (33) Google Scholar, 28Yoo O.-J. Yoon H.-S. Beak K.-H. Jeon C.-J. Miyamoto K. Ueno A. Agarwal K. Nucleic Acids Res. 1991; 19: 1073-1079Crossref PubMed Scopus (41) Google Scholar). Yeast S-II specifically stimulated yeast RNA polymerase II (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar) and was shown to promote cleavage and elongation of nascent RNA in the elongation complex of transcription in vitro (29Christie K.R. Awrey D.E. Edwards A.M. Kane C.M. J. Biol. Chem. 1994; 269: 936-943Abstract Full Text PDF PubMed Google Scholar, 30Johnson T.L. Chamberlin M.J. Cell. 1994; 77: 217-224Abstract Full Text PDF PubMed Scopus (64) Google Scholar). Therefore, like the S-IIs of higher eukaryotes, yeast S-II is a transcription elongation factor. The nucleotide sequence of the yeast S-II gene indicated that it is the same protein as STPα and the product of the PPR2/DST1 gene. The PPR2/DST1 gene was originally identified as the gene responsible for 6-azauracil sensitivity of yeast (31Hubert J.-C. Guyonvarch A. Kammerer B. Exinger F. Liljelund P. Lacroute F. EMBO J. 1983; 2: 2071-2073Crossref PubMed Scopus (50) Google Scholar). STPα catalyzes the transfer of a strand from a duplex linear molecule of DNA to a complementary circular single strand (32Sugino A. Nitiss J. Pesnick M.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3683-3687Crossref PubMed Scopus (47) Google Scholar, 33Clark A.B. Dykstra C.C. Sugino A. Mol. Cell. Biol. 1991; 11: 2576-2582Crossref PubMed Google Scholar), indicating that this protein has pleiotropic functions. On the other hand, gene disruption experiments revealed that the S-II null mutant is viable (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar, 31Hubert J.-C. Guyonvarch A. Kammerer B. Exinger F. Liljelund P. Lacroute F. EMBO J. 1983; 2: 2071-2073Crossref PubMed Scopus (50) Google Scholar, 33Clark A.B. Dykstra C.C. Sugino A. Mol. Cell. Biol. 1991; 11: 2576-2582Crossref PubMed Google Scholar) but that the mutation induces sensitivity to 6-azauracil (31Hubert J.-C. Guyonvarch A. Kammerer B. Exinger F. Liljelund P. Lacroute F. EMBO J. 1983; 2: 2071-2073Crossref PubMed Scopus (50) Google Scholar). It is not known why the S-II null mutant is sensitive to 6-azauracil. However, 6-azauracil sensitivity is the only prominent phenotype of the S-II null mutant, and the function of S-II as a transcription factor may be indispensable for suppressing 6-azauracil sensitivity. To gain more insight into the structure-function relationship of S-II, we created various deletion mutants of S-II and examined their abilities to stimulate RNA polymerase II, relieve arrest, and suppress the 6-azauracil sensitivity of the S-II null mutant. Results indicated that all of these activities are due to the same motif in the S-II molecule, suggesting that 6-azauracil sensitivity is caused by loss of function of S-II as a transcription factor. The S-II null mutant was established previously from ANY102 (Mata/Matα, leu2/leu2, his/his, trp1/trp1, ura3/ura3) by inserting the LEU2 gene into the S-II gene (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar). Therefore, the null mutant required uracil and could not be used for assay of suppression of 6-azauracil sensitivity. So, we established a new S-II null mutant by inserting the URA3 gene. Gene disruption method used was essentially as reported before (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar). Briefly, pBSM13 carrying a 4.0-kilobase fragment of YSII::LEU2 was digested with XbaI and ClaI to remove the LEU2 gene, and the URA3 gene was ligated instead of the LEU2 gene. The insert was purified and used for transformation of TNY04 obtained by tetrad dissection of ANY102 (34Gietz D. Jean A.S. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2895) Google Scholar). One of the URA+ transformants thus obtained was named TNY14. Disruption of the S-II gene in TNY14 was confirmed by Southern blot analysis as described before (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar). Yeast cells were transformed essentially as described by Gietz et al. (34Gietz D. Jean A.S. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2895) Google Scholar). They were grown in 5 ml of YPD medium (1% polypeptone, 1% yeast extract, 2% glucose) at 30°C to an optical density at 600 nm (A600) of 2.0 and then collected and suspended in 50 μl of TELiAc (10 m M Tris/HCl buffer, pH 7.5, 1 m M EDTA, 100 m M CH3COOLi). This suspension was incubated with 100 ng of plasmids, 50 μg of heat-denatured calf thymus DNA, and 300 μl of TELiAc containing 40% polyethylene glycol 4000 for 30 min at 30°C and then for 20 min at 42°C. The cells were collected, suspended in sterilized water, and spread on YNBD plates (0.67% yeast nitrogen base without amino acids, 2% glucose), supplemented with 20 μg/ml each of adenine sulfate, tryptophan, histidine, and 30 μg/ml of leucine, and incubated for 3 days at 30°C. Plasmids containing S-II deletion mutants were constructed as follows. S-II deletion mutants were amplified by polymerase chain reaction using the S-II gene (pYSII-2) as a template, and the resulting DNA fragments were ligated to the GAL1 promoter in pYO324. Mutant S-II proteins were induced by galactose. Transformed cells were cultured in EMD medium (0.67% yeast nitrogen base without amino acids, 0.5% casamino acids technical, 2% glucose) with an appropriate supplement(s) at 30°C until A600 reached about 2.0. Then 2.5 × 106cells were transferred to 0.5 ml of fresh medium and incubated at 30°C for 2 h. The cell suspension was diluted 1000-fold with sterilized water, and 120 μl of the diluted cell suspension was spread on YNBD or YNBGS (0.67% yeast nitrogen base without amino acids, 5% galactose, and 0.2% sucrose) plates containing an appropriate supplement(s) with or without 100 μg/ml 6-azauracil. Colonies on YNBGS plates were examined after incubation at 30°C for 5 days. This assay was done essentially as described before in the presence and absence of each recombinant S-II deletion mutant protein (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar). The reaction mixture (60 μl) contained 50 m M Tris/HCl (pH 7.9), 1.6 m M MnCl2, 0.5 m M each of ATP, GTP, and CTP, 0.01 m M UTP, 18.5 kBq of [3H]UTP, 10 m M 2-mercaptoethanol, 2 μg of calf thymus DNA, and 8 units of partially purified S. cerevisiae RNA polymerase II. The reaction mixture was incubated for 20 min at 30°C, and then the radioactivity incorporated into the acid-insoluble fraction was counted. 1 unit of RNA polymerase II was defined as the amount incorporating 1 pmol of UMP into the acid-insoluble fraction. This assay was done essentially as described by Christie et al. (29Christie K.R. Awrey D.E. Edwards A.M. Kane C.M. J. Biol. Chem. 1994; 269: 936-943Abstract Full Text PDF PubMed Google Scholar), using a 3′-deoxycytidine-extended template (35Kadesch T.R. Chamberlin M.J. J. Biol. Chem. 1982; 257: 5286-5295Abstract Full Text PDF PubMed Google Scholar) of the TaqI fragment containing the human histone H3.3 gene (36Reines D. Wells D. Chamberlin M.J. Kane C.M. Mol. Cell. Biol. 1987; 196: 299-312Google Scholar), RNA polymerase II, and S-II mutant proteins. The dC-tailed template used was kindly provided by Dr. C. M. Kane (University of California, Berkeley Dept. of Molecular and Cell Biology). Recombinant S-II mutant proteins were expressed in Escherichia coli using the T7 expression system, as described before (37Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4834) Google Scholar). Plasmids containing S-II deletion mutants were constructed as follows. S-II deletion mutants were amplified by polymerase chain reaction using pYSII-2 as a template, and the resulting DNA fragments were ligated into an expression vector, pET-3d. The resulting plasmids were transfected into E. coli BL21(DE3)/pLysE, and induction of S-II mutant proteins was performed by treating the E. coli cells with isopropyl-1-thio-β- D-galactopyranoside. Recombinant S-II mutant proteins were purified as follows. Freshly harvested E. coli cells were lysed by treatment with lysozyme-EDTA and deoxycholate, and the lysate was centrifuged at 100,000 × g for 1 h. The resulting supernatant was diluted with buffer 1 (50 m M Tris/HCl, pH 7.9, 5 m M 2-mercaptoethanol, 0.1% Triton X-100) and then subjected to fast protein liquid chromatography on a Mono-S HR 5/5 column equilibrated with buffer 1. Recombinant S-II deletion mutant proteins were eluted with buffer 1 containing 0.05-0.2 M NaCl. The fraction of Δ3-151 protein from the Mono-S column was further purified on a column of Superose 12 with buffer 1 containing 1 M NaCl. Recombinant S-II mutant proteins were detected by immunoblotting with antibody against yeast S-II. S-II and partially purified RNA polymerase II from S. cerevisiae were prepared as described before (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar). Antibody against S-II was raised by injecting purified S-II into male albino rabbits. On immunoblotting, this antibody detected S-II in the crude extract of yeast as a single band. DNA manipulations including restriction enzyme digestion, gel electrophoresis, DNA ligation, plasmid isolation, and E. coli transformation were carried out by standard methods. SDS-polyacrylamide gel electrophoresis and immunoblot analysis were performed as described before (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar, 39Homma K. Kurata S. Natori S. J. Biol. Chem. 1994; 269: 15258-15264Abstract Full Text PDF PubMed Google Scholar). Protein was determined by the method of Bradford (40Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar). Previous studies showed that S-II, the PPR2/DST1 gene product, and STPα are almost certainly the same protein (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar, 31Hubert J.-C. Guyonvarch A. Kammerer B. Exinger F. Liljelund P. Lacroute F. EMBO J. 1983; 2: 2071-2073Crossref PubMed Scopus (50) Google Scholar, 33Clark A.B. Dykstra C.C. Sugino A. Mol. Cell. Biol. 1991; 11: 2576-2582Crossref PubMed Google Scholar). We are interested in the functional motifs in the S-II molecule. To examine whether the region needed for stimulation of RNA polymerase II and that needed for 6-azauracil sensitivity in the S-II molecule could be separated, we constructed plasmids carrying the genes for various S-II deletion mutant proteins and used these plasmids for in vivo and in vitro experiments. We examined the ability of S-II deletion mutant proteins to suppress 6-azauracil sensitivity by introducing these plasmids into the yeast S-II null mutant and expressing the mutant proteins in vivo. We also expressed these plasmids in E. coli, isolated mutant S-II proteins, and examined their ability to stimulate yeast RNA polymerase II in vitro. The S-II null mutants that we established previously required uracil (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar), so they could not be used for testing suppression of 6-azauracil sensitivity. Therefore, we constructed a new S-II null mutant. Southern blot analysis of genomic DNA derived from TNY14 showed that its S-II gene was disrupted. We further confirmed that S-II is not present in TNY14 by an immunofluorescence study with affinity-purified anti-S-II antibody. Immunofluorescence was exclusively detected in the nuclei of wild type yeast, whereas no immunofluorescence was detected in TNY14 (data not shown). Thus, we concluded that S-II is a nuclear protein and that TNY14 lacks this protein. TNY14 was sensitive to 6-azauracil and did not form colonies on MVD agar plates containing 100 μg/ml of 6-azauracil (Fig. 1, A and B). When pYSG6 (an expression vector containing the GAL1 promoter-yeast S-II gene coding region) was introduced into TNY14, the resulting transformant could form colonies on MVGS agar plates in the presence of 6-azauracil (Fig. 1, C and D), indicating that introduction of full-length S-II cDNA suppressed the 6-azauracil sensitivity of TNY14. However, it did not form colonies on MVD plates as expected. Using this system, we examined the suppressions of 6-azauracil sensitivity by various S-II deletion mutants. The amino acid sequence of S-II is known to be conserved in various eukaryotes. In particular, about 50 residues in the carboxyl-terminal region are highly conserved (about 70% similarity), and about 80 residues in the amino-terminal region are also relatively well conserved (about 38% similarity) (10Hirashima S. Hirai H. Nakanishi Y. Natori S. J Biol. Chem. 1988; 263: 3858-3863Abstract Full Text PDF PubMed Google Scholar, 13Kanai A. Kuzuhara T. Sekimizu K. Natori S. J. Biochem. 1991; 109: 674-677Crossref PubMed Scopus (25) Google Scholar, 15Xu Q. Nakanishi T. Sekimizu K. Natori S. J. Biol. Chem. 1994; 269: 3100-3103Abstract Full Text PDF PubMed Google Scholar, 26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar, 27Marshall T.K. Guo H. Price D.H. Nucleic Acids Res. 1990; 18: 6293-6298Crossref PubMed Scopus (33) Google Scholar, 28Yoo O.-J. Yoon H.-S. Beak K.-H. Jeon C.-J. Miyamoto K. Ueno A. Agarwal K. Nucleic Acids Res. 1991; 19: 1073-1079Crossref PubMed Scopus (41) Google Scholar). Christie et al. (29Christie K.R. Awrey D.E. Edwards A.M. Kane C.M. J. Biol. Chem. 1994; 269: 936-943Abstract Full Text PDF PubMed Google Scholar) demonstrated that in yeast S-II, lacking the first 113 residues has the same activity as the full-length form to read-through the pausing site. On the bases of these results, we constructed seven clones of S-II deletion mutants. Of these, five were deletion mutants of the amino-terminal side, and two were those of the carboxyl-terminal side. We found that the amino-terminal mutants Δ2-123 (in which residues 2-123 were deleted), Δ2-141, and Δ3-147 suppressed the 6-azauracil sensitivity of TNY14 but that Δ2-149 and Δ2-151 did not. These results indicated that residues 1-147, including the relatively well conserved sequence in the amino-terminal region, are not essential for suppression of 6-azauracil sensitivity. The carboxyl-terminal mutant, Δ260-309, did not suppress 6-azauracil sensitivity, indicating that the 49 carboxyl-terminal residues are necessary for suppressing 6-azauracil sensitivity. We confirmed the expression of each deletion mutant protein in the corresponding transformant by immunoblot analysis, as shown in Fig. 2. We then expressed these S-II deletion mutants in E. coli, purified each protein to near homogeneity, and examined its ability to stimulate RNA polymerase II and relieve arrest in vitro. As shown in Fig. 3 A, each protein gave essentially a single band on SDS-polyacrylamide gel electrophoresis. Immunoblotting again showed that all the mutant proteins reacted with antibody against S-II (Fig. 3 B). As is evident from Fig. 4, wild type S-II and the Δ2-123 protein had almost the same stimulatory activity, but the Δ2-141 protein had less, and the Δ2-151 protein had none. Therefore, it is clear that the first 141 residues are not essential for stimulation of RNA polymerase II. As the Δ266-309 protein had no stimulatory activity, the last 44 residues in the carboxyl-terminal region are essential for stimulation of RNA polymerase II.FIG. 4Stimulation of RNA polymerase II by S-11 deletion mutant proteins. Increasing amounts of purified recombinant S-II mutant proteins were added to 8 units of partially pU!;fied yeast RNA polymerase II, and their activity in stimulating RNA polymerase II was measured under standard assay conditions. Lines indicate activity of mutant proteins and S-II (wt) as shown on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also examined arrest-relief activity of these mutant proteins. As shown in Fig. 5, wild type S-II, Δ2-123, and Δ2-141 proteins promoted read-through by RNA polymerase II at specific blocks to elongation in the human histone H3.3 gene, sites designated TIa, TIb, and TII (36Reines D. Wells D. Chamberlin M.J. Kane C.M. Mol. Cell. Biol. 1987; 196: 299-312Google Scholar, 41Kerpolla T.K. Kane C.M. Biochemistry. 1990; 29: 269-278Crossref PubMed Scopus (82) Google Scholar). However, Δ3-151 and Δ266-309 proteins had no appreciable activity to relieve transcription arrest at these sites. Summary of two independent arrest-relief experiments is shown in Table I. These results strongly suggested that the ability to stimulate RNA polymerase II and to relieve arrest at specific pausing sites are the same activity, and that this activity is essential for suppression of 6-azauracil sensitivity of S-II null mutants, as summarized in Fig. 6.Table I:Summary of activities of S-II mutant proteins to promote read-through of RNA polymerase IITable I:Summary of activities of S-II mutant proteins to promote read-through of RNA polymerase IIData were obtained from the quantitation of the read-through experiments described in the legend to Fig. 5. Percentage radioactivity of run-off transcript (RO) or RNA stalled at TIa/total radioactivity (RO + TIa) was determined by counting the radioactivity in each band in Fig. 5. Each value represents the average of two experiments with S.D.FIG. 6Schematic presentation of S-11 deletion mutants and their activity. Open boxes represent protein coding regions. Names of deletion mutants are given on the left of each box with deleted amino acid residues. The highly conserved region in the carboxyl-tenninal region and relatively conserved region in the amino-tenninal region are indicated by arrows with percentage similarities between yeast and Ehrlich cell S-II. Activities are shown as Yes or No. n.d., not detennined; a.a., amino acids.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Data were obtained from the quantitation of the read-through experiments described in the legend to Fig. 5. Percentage radioactivity of run-off transcript (RO) or RNA stalled at TIa/total radioactivity (RO + TIa) was determined by counting the radioactivity in each band in Fig. 5. Each value represents the average of two experiments with S.D. Using various S-II deletion mutants of yeast, we demonstrated that at least 1-147 residues from the amino-terminal are not essential, and the last 162 residues are sufficient for suppression of the 6-azauracil sensitivity of an S-II null mutant. We also showed that for activity to stimulate RNA polymerase II and relieve arrest, there is a critical point in S-II between residues 142 and 151, and that 168 but not 158 of the carboxyl-terminal residues were necessary for stimulation of RNA polymerase II and relief of transcription arrest in vitro. Therefore, this region should contain binding regions for both RNA polymerase II and nucleic acid (6Horikoshi M. Sekimizu K. Natori S. J. Biol. Chem. 1984; 259: 608-611Abstract Full Text PDF PubMed Google Scholar, 42Agarwal K. Beak K.-H. Jeon C.-J. Miyamoto K. Ueno A. Yoon H.-S. Biochemistry. 1991; 30: 7842-7851Crossref PubMed Scopus (71) Google Scholar, 43Quian X. Joen C.-J. Yoon H.-S. Agarwal K. Weiss M.A. Nature. 1993; 365: 277-279Crossref PubMed Scopus (111) Google Scholar). For technical reasons, we could not purify the Δ2-147 and Δ2-149 proteins in an E. coli lysate and thus could not examine their abilities to stimulate RNA polymerase II and relieve arrest. Nonetheless, it is quite clear that there is a good correlation between the activity for suppression of 6-azauracil sensitivity in vivo and that for stimulation of RNA polymerase II (and thus for arrest relief) in vitro, and that nearly half of the 309 residues of S-II are not directly related to these activities. Christie et al. (29Christie K.R. Awrey D.E. Edwards A.M. Kane C.M. J. Biol. Chem. 1994; 269: 936-943Abstract Full Text PDF PubMed Google Scholar) also reported that there is essentially no difference in the cleavage or read-through activities between complete yeast S-II and a mutant S-II lacking the first 113 residues. Probably, the functional motif of S-II is in the carboxyl-terminal half, and the amino-terminal half is the regulatory motif, since this region of S-II of Ehrlich cell has been shown to contain phosphorylation sites (7Horikoshi M. Sekimizu K. Hirashima S. Mitsuhashi Y. Natori S. J. Biol. Chem. 1985; 260: 5739-5744Abstract Full Text PDF PubMed Google Scholar). However, read-through mechanism of S-II may not be so simple because Cipres-Palacin and Kane (44Cipres-Palacin G. Kane C.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8087-8091Crossref PubMed Scopus (38) Google Scholar) recently reported S-II mutants that are inactive for promoting read-through, although they stimulated cleavage of the nascent transcript in stalled elongation complexes (44Cipres-Palacin G. Kane C.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8087-8091Crossref PubMed Scopus (38) Google Scholar). Unlike mutants lacking regions of the amino-terminal half, those lacking about 50 residues from the carboxyl-terminal were shown to have lost all the stimulatory, arrest-relief, and suppressive activities. Again, we could not examine these activities with a single mutant for technical reasons, but we deduced that the Δ260-309 protein had no stimulatory and arrest-relief activity from the fact that the 6-residue-longer Δ266-309 protein had no activity. These genetical and biochemical results strongly suggest that 6-azauracil sensitivity is due to loss of function of S-II as a transcription factor. There seem to be two possible reasons why the S-II null mutant has acquired the phenotype of 6-azauracil sensitivity. One is that S-II is essential for the transcription of another gene that causes 6-azauracil sensitivity, and the S-II null mutant lacks this gene product. This gene product may not be essential for the growth of yeast, since the S-II null mutant is not lethal (26Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar, 31Hubert J.-C. Guyonvarch A. Kammerer B. Exinger F. Liljelund P. Lacroute F. EMBO J. 1983; 2: 2071-2073Crossref PubMed Scopus (50) Google Scholar, 33Clark A.B. Dykstra C.C. Sugino A. Mol. Cell. Biol. 1991; 11: 2576-2582Crossref PubMed Google Scholar). The other possibility is that many pausing sites are introduced into the yeast genome in the presence of 6-azauracil, and the S-II null mutant becomes lethal in the presence of 6-azauracil, as these pausing sites cannot be read-through by RNA polymerase II in the absence of S-II. With respect to the latter possibility, Exinger and Lacroute (45Exinger F. Lacroute F. Curr. Genet. 1992; 22: 9-11Crossref PubMed Scopus (215) Google Scholar) reported that 6-azauracil inhibits GTP synthesis in S. cerevisiae and also causes significant decrease in the UTP content. In vitro transcription experiments showed that reduction of at least one of four nucleoside triphosphates in the reaction mixture induced pause of transcription elongation and that addition of S-II released this cessation of transcription elongation (18Izban M.G. Luse D.S. Genes & Dev. 1992; 6: 1342-1356Crossref PubMed Scopus (227) Google Scholar, 20Reines D. J. Biol. Chem. 1992; 267: 3795-3800Abstract Full Text PDF PubMed Google Scholar, 21Reines D. Ghanouni P. Li Q.-q Mote Jr., J. J. Biol. Chem. 1992; 267: 15516-15522Abstract Full Text PDF PubMed Google Scholar, 29Christie K.R. Awrey D.E. Edwards A.M. Kane C.M. J. Biol. Chem. 1994; 269: 936-943Abstract Full Text PDF PubMed Google Scholar). Therefore, it is possible that many pausing sites are created in various class II genes and that their transcriptions are inhibited in the presence of 6-azauracil, interfering with the growth of the S-II null mutant (46Archambault J. Lacroute F. Ruet A. Friesen J.D. Mol. Cell. Biol. 1992; 12: 4142-4152Crossref PubMed Scopus (120) Google Scholar). If the former possibility is the case, there should be a specific gene(s) responsible for 6-azauracil sensitivity that is transcribed only in the presence of S-II. Identification and isolation of this gene(s) may give a clue to the function of S-II in vivo. If the latter possibility is correct, this S-II null mutant should become lethal when the nucleoside triphospate pool in the cells is reduced by other methods. It is unlikely that S-II itself has activity to detoxify 6-azauracil or to disturb the uptake of 6-azauracil, thus making the S-II null mutant sensitive to 6-azauracil, because, in general, metabolic enzymes are present in the cytoplasm and barrier proteins are present in the membrane, whereas S-II was found almost exclusively in the nuclei. We thank Dr. Caroline M. Kane for supplying the template and protocol for arrest-relief assay.
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