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Cleavage, but Not Read-through, Stimulation Activity Is Responsible for Three Biologic Functions of Transcription Elongation Factor S-II

2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês

10.1074/jbc.m211384200

1083-351X

Toshiharu Ubukata, Tomoko Shimizu, Nobuaki Adachi, Kazuhisa Sekimizu, Toshiyuki Nakanishi,

Genomics and Chromatin Dynamics

Transcription elongation factor S-II stimulates cleavage of nascent transcripts generated by RNA polymerase II stalled at transcription arrest sites. In vitro experiments have shown that this action promotes RNA polymerase II to read through these transcription arrest sites. This S-II-mediated cleavage is thought to be necessary, but not sufficient, to promote read-through in thein vitro systems. Therefore,Saccharomyces cerevisiae strains expressing S-II mutant proteins with different in vitro activities were used to study both the cleavage and the read-through stimulation activities of S-II to determine which S-II functions are responsible for its biologic functions. Strains expressing mutant S-II proteins active in both cleavage and read-through stimulation were as resistant as wild type strains to 6-azauracil and mycophenolic acid. 6-Azauracil also inducedIMD2 gene expression in both these mutant strains and the wild type. Furthermore, strains having a genotype consisting of one of these S-II mutations and the spt4 null mutation grew as well as the spt4 null mutant at 37 °C, a restrictive temperature for a strain bearing double null mutations ofspt4 and S-II. In contrast, strains bearing S-II mutations defective in both cleavage and read-through stimulation had phenotypes similar to those of an S-II null mutant. However, one strain expressing a mutant S-II protein active only in cleavage stimulation had a phenotype similar to that of the wild type strain. These results suggest that cleavage, but not read-through, stimulation activity is responsible for all three biologic functions of S-II (i.e.suppression of 6-azauracil sensitivity, induction of theIMD2 gene, and suppression of temperature sensitivity ofspt4 null mutant). Transcription elongation factor S-II stimulates cleavage of nascent transcripts generated by RNA polymerase II stalled at transcription arrest sites. In vitro experiments have shown that this action promotes RNA polymerase II to read through these transcription arrest sites. This S-II-mediated cleavage is thought to be necessary, but not sufficient, to promote read-through in thein vitro systems. Therefore,Saccharomyces cerevisiae strains expressing S-II mutant proteins with different in vitro activities were used to study both the cleavage and the read-through stimulation activities of S-II to determine which S-II functions are responsible for its biologic functions. Strains expressing mutant S-II proteins active in both cleavage and read-through stimulation were as resistant as wild type strains to 6-azauracil and mycophenolic acid. 6-Azauracil also inducedIMD2 gene expression in both these mutant strains and the wild type. Furthermore, strains having a genotype consisting of one of these S-II mutations and the spt4 null mutation grew as well as the spt4 null mutant at 37 °C, a restrictive temperature for a strain bearing double null mutations ofspt4 and S-II. In contrast, strains bearing S-II mutations defective in both cleavage and read-through stimulation had phenotypes similar to those of an S-II null mutant. However, one strain expressing a mutant S-II protein active only in cleavage stimulation had a phenotype similar to that of the wild type strain. These results suggest that cleavage, but not read-through, stimulation activity is responsible for all three biologic functions of S-II (i.e.suppression of 6-azauracil sensitivity, induction of theIMD2 gene, and suppression of temperature sensitivity ofspt4 null mutant). 6-azauracil mycophenolic acid Transcription elongation factor S-II, originally purified from mouse Ehrlich ascites tumor cells, is an RNA polymerase II-stimulating factor in promoter-independent RNA synthesis (1Sekimizu K. Nakanishi Y. Mizuno D. Natori S. Biochemistry. 1979; 18: 1582-1588Google Scholar). It has been found in all eukaryotes thus far investigated, and its primary structure is highly conserved (2Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Google Scholar, 3Hirashima S. Hirai H. Nakanishi Y. Natori S. J. Biol. Chem. 1988; 263: 3858-3863Google Scholar, 4Xu Q. Nakanishi T. Sekimizu K. Natori S. J. Biol. Chem. 1994; 269: 3100-3103Google Scholar, 5Chen H.C. England L. Kane C.M. Gene (Amst.). 1992; 116: 253-258Google Scholar, 6Sluder A.E. Greenleaf A.L. Price D.H. J. Biol. Chem. 1989; 264: 8963-8969Google Scholar, 7Taira Y. Kubo T. Natori S. J. Biol. Chem. 2000; 275: 32011-32015Google Scholar). S-II is a unique transcription elongation factor that promotes transcript elongation through transcription arrest sites found in genes (8Reinberg D. Roeder R.G. J. Biol. Chem. 1987; 262: 3331-3337Google Scholar, 9Reines D. Chamberlin M.J. Kane C.M. J. Biol. Chem. 1989; 264: 10799-10809Google Scholar, 10SivaRaman L. Reines D. Kane C.M. J. Biol. Chem. 1990; 265: 14554-14560Google Scholar, 11Christie K.R. Awrey D.E. Edwards A.M. Kane C.M. J. Biol. Chem. 1994; 269: 936-943Google Scholar). The molecular mechanism of this phenomenon has been investigated extensively in in vitrosystems, and it has been shown that S-II stimulates the nuclease activity of RNA polymerase II, which then cleaves the nascent transcript. Then the 3′-end of the nascent RNA is realigned with the catalytic site of RNA polymerase II, and the transcription elongation complex tries reading through the arrest site again (11Christie K.R. Awrey D.E. Edwards A.M. Kane C.M. J. Biol. Chem. 1994; 269: 936-943Google Scholar, 12Izban M.G. Luse D.S. Genes Dev. 1992; 6: 1342-1356Google Scholar, 13Reines D. J. Biol. Chem. 1992; 267: 3795-3800Google Scholar, 14Reines D. Ghanouni P. Gu W. Mote Jr., J. Powell W. Cell Mol. Biol. Res. 1993; 39: 331-338Google Scholar). The cleavage stimulation activity of S-II can be separated from its read-through stimulating activity; although the cleavage stimulation activity is essential, it is not sufficient to promote read-through of RNA polymerase II in vitro (15Cipres-Palacin G. Kane C.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8087-8091Google Scholar). There have been several reports describing the function of S-II inSaccharomyces cerevisiae. In several yeast strains bear null mutations of the genes encoding the transcription elongation machinery, such as spt4Δ, rpb9Δ, and ctk1Δ, S-II is indispensable for cell proliferation under certain culture conditions (16Davie J.K. Kane C.M. Mol. Cell. Biol. 2000; 20: 5960-5973Google Scholar, 17Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Google Scholar, 18Jona G. Wittschieben B.O. Svejstrup J.Q. Gileadi O. Gene. 2001; 267: 31-36Google Scholar). Although Kulish et al. (19Kulish D. Struhl K. Mol. Cell. Biol. 2001; 21: 4162-4168Google Scholar) reported that S-II promotes read-through of RNA polymerase II in vivoas well as in vitro, it remains to be elucidated whether S-II functions in vivo through its cleavage-stimulating activity, its read-through-stimulating activity, or both. S-II also plays a role in the development of 6-AU1 or MPA resistance in yeast (2Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Google Scholar, 20Exinger F. Lacroute F. Curr. Genet. 1992; 22: 9-11Google Scholar) and in the expression of the pyrimidine-specific 5′-nucleotidase SDT1 gene; it is also involved in the induction of the IMP dehydrogenase IMD2 gene and the polyamine transporter TPO1 gene in response to treatment with 6-AU or MPA (21Shimoaraiso M. Nakanishi T. Kubo T. Natori S. J. Biol. Chem. 2000; 275: 29623-29627Google Scholar, 22Shaw R.J. Reines D. Mol. Cell. Biol. 2000; 20: 7427-7437Google Scholar, 23Desmoucelles C. Pinson B. Saint-Marc C. Daignan-Fornier B. J. Biol. Chem. 2002; 277: 27036-27044Google Scholar). Stimulation of expression of these genes is thought to be necessary for developing 6-AU and MPA resistance inS. cerevisiae. In this study, the two S-II activities were investigated in an effort to determine which are responsible for the biologic functions, such as drug resistance and transcription induction, in S. cerevisiae. Site-directed mutations were introduced into the S-II gene to generate mutant yeast strains that express S-II mutant proteins with different levels of cleavage and read-through stimulation activities in vitro. These strains were then tested to determine their sensitivity to 6-AU and MPA, their ability to induce the IMD2 gene in response to 6-AU treatment, and their temperature-sensitive growth retardation in combination with thespt4 null mutation. The results show that cleavage-stimulating, but not read-through-stimulating, activity correlates with the biologic functions of S-II, suggesting that the former activity of S-II is responsible for its functions in vivo. All yeast strains used in this study are summarized in Table I. HKY01 and HKY02 are S-II null mutant strains (courtesy of Dr. T. Ito) derived from YPH499 (24Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Google Scholar); S-II gene was disrupted by URA3 orHIS3 gene introduction in these strains, respectively. Plasmid pH1180 was constructed by inserting the 3.4-kb PvuII fragment of the S. cerevisiae S-II gene into pBluescript KS(+) (Stratagene, La Jolla, CA). To screen for mutant clones, silent base-exchange mutations were introduced to form the following new restriction enzyme recognition sites in each mutant: KasI sites for Mt1 and Mt2; SacII sites for Mt3, Mt4, Mt8, and Mt9; an SphI site for Mt5; a BspT104I site for Mt6; and a PvuI site for Mt7. pMt1b was constructed by inserting the PstI-NcoI fragment of the S-II gene amplified by PCR with the primers 5′-AAACTGCAGGATCTGGCGCCAGCGCCCTTAGCTGCAAAGATAGAAG-3′ and 5′-GGGGCCAGTTGTTCAGGTCCTAACTGTATGCC-3′ (TU-23) into thePstI-NcoI site of pH1180. pMt1 was constructed by inserting the PstI-KasI fragment of the S-II gene amplified by PCR with the primers 5′-CACAGTGTAGTCAGTCCGCATAAGAGCATTCATCATGG-3′ (TU-22) and 5′-TAAGGGCGCTGGCGCCAGATCCTTGGCATCG-3′ into thePstI-KasI site of pMt1b. pMt2b was constructed by inserting the PstI-NcoI fragment of the S-II gene amplified by PCR with the primers TU-22 and 5′-CATGCCATGGTGGCGCCAGCTGCGGCGTATAAGGCTTGCTTGGC-3′ into thePstI-NcoI site of pH1180. pMt2 was constructed by inserting the KasI-NcoI fragment of the S-II gene amplified by PCR with the primers 5′-CAACGCACAGGGCGCCACCATAGAAAGG-3′ and TU-23 into the KasI-NcoI site of pMt2b. pMt3b was constructed by inserting the MfeI-NcoI fragment of the S-II gene amplified by PCR with the primers 5′-CTATCAATTGCAAACACAATCCGCGGATTTGCCATTGACCAC-3′ and TU-23 into theMfeI-NcoI site of pYSII-2 (2Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Google Scholar). pMt3 was constructed by inserting the PstI-NcoI fragment of pYSII-2 to the PstI-NcoI site of pH1180. pMt4b was constructed by inserting the PstI-NcoI fragment of the S-II gene amplified by PCR with the primers 5′-AAACTGCAGATCCGCGGATGAACCATTGACCACTGCTTGTACATGTG-3′ and TU-23 into the PstI-NcoI site of pH1180. pMt4 was constructed by inserting the PstI-SacII fragment of the S-II gene amplified by PCR with the primers TU-22 and 5′-GGTTCATCCGCGGATCTTGTTTGCAATTG-3′ into thePstI-SacII site of pMt4b. pMt5b was constructed by inserting the PstI-NcoI fragment of the S-II gene amplified by PCR with the primers 5′-AAACTGCAGAAGCATGCGGTAACAGATGGGCTTTCTCTTAGAATAG-3′ and TU-23 into the PstI-NcoI site of pH1180. pMt5 was constructed by inserting the PstI-SphI fragment of the S-II gene amplified by PCR with the primers TU-22 and 5′-CATCTGTTACCGCATGCTTCACATGTACAG-3′ into thePstI-SphI site of pMt5. pMt6 to -9 were obtained by using a QuikChange site-directed mutagenesis kit (Stratagene) and pH1180 as a template. Primers used for mutants were 5′-GCCAGGTATGCTATAATTTATTCGAACGTCATATC-3′ and 5′-GATATGACGTTCGAATAAATTATAGCATACCTGGC-3′ for pMt6, 5′-CAGTCACCGATCGAGCTACATGTGGTAAATG-3′ and 5′-CATTTACCACATGTAGCTCGATCGGTGACTG-3′ for pMt7, 5′-CAATTGCAAACACAATCCGCGGATAATCCATTGACC-3′ and 5′-GGTCAATGGATTATCCGCGGATTGTGTTTGCAATTG-3′ for pMt8, and 5′-CAATTGCAAACACAATCCGCGGATGAACC-3′ and 5′-GGTTCATCCGCGGATTGTGTTTGCAATTG-3′ for pMt9. After the presence of the mutations on the plasmid clones was confirmed by DNA sequencing, the BamHI-PvuI (pMt2) or PvuII (all other constructs) fragments were excised, and an EZ Yeast Transformation Kit (Qbiogene, Inc., Carlsbad, CA) was used to introduce the fragments separately into HKY01 to create the S-II mutant strains designated Mt1 to Mt9. When the mutant S-II fragment is introduced by homologous recombination into the S-II locus, it loses theURA3 gene instead and is transformed to 5-fluoroorotic acid-resistant. Therefore, we isolated 5-fluoroorotic acid-resistant HKY01 transformants and performed genomic PCR to screen for the desired mutants with the primers 5′-CACTCGATGATGGGACTACG-3′ and 5′-GCGCTAGTAAGACAGATAGG-3′ followed by restriction enzyme digestion. The introduction of the mutant sequences into the S-II locus was confirmed by Southern blot analysis. S-II genes were amplified from each S-II mutant strain, and sites of mutation were confirmed by DNA sequencing. Each mutant strain bore only the introduced mutation.Table IYeast strains used in this studyStrainGenotypeHKY01MATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1::URA3HKY02MATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1::HIS3TU100MATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 [pRS416 (URA3 CEN)]TU101MATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 [pRS416 (URA3 CEN)] [pRS413 (HIS3 CEN)]TU102MATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1::HIS3 [pRS416 (URA3 CEN)]Mt1MATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1K242A/Q243AMt2MATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1N252A/N255A/Q257AMt3MATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1R287Q/E291LMt4MATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1F296AMt5MATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1K307AMt6MATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1R200AMt7MATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1F269AMt8MATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1R287Q/E291NMt9MATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1R287Qspt4ΔMATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 spt4::URA3dst1Δspt4ΔMATa ura3–52 lys2–801amberade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1::HIS3 spt4::URA3Mt1spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1K242A/Q243A spt4::URA3Mt2spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1N252A/N255A/Q257A spt4::URA3Mt3spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1R287Q/E291L spt4::URA3Mt4spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1F296A spt4::URA3Mt5spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1K307A spt4::URA3Mt6spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1R200A spt4::URA3Mt7spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1F269A spt4::URA3Mt8spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1R287Q/E291N spt4::URA3Mt9spt4ΔMATa ura3–52 lys2–801amber ade2–101ochre trp1–Δ63 his3-Δ200 leu2-Δ1 dst1R287Q spt4::URA3 Open table in a new tab A PCR-based gene disruption method (25Burke D. Dawson D. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 139-140Google Scholar) was used to prepare allspt4Δ strains. PCR with the plasmid pRS416 (Stratagene) as a template was used to prepare the DNA fragment containing the URA3 gene used to disrupt the SPT4gene. The primers used were 5′-ATTCATTACTATTATACATGTGATATCAGAACGGAAGGTTAGATTGTACTGAGAGTGCAC-3′ and 5′-TTACACCTGGCCACATTCAGTTTGGCAAAAGCGAACGAGGCTGTGCGGTATTTCACACCG-3′. The resulting fragment was separately introduced to YPH499, HKY02, and Mt1 to -9 using an EZ Yeast Transformation Kit.URA + transformants were selected on SD (ura−) agar plates (0.67% (w/v) yeast nitrogen base without amino acids; 2% (w/v) glucose; 0.1 mg/ml each histidine, adenine sulfate, tryptophan, and lysine; and 0.25 mg/ml leucine; 2% bactoagar), and Southern blot analysis was used to confirm gene disruption. Recombinant yeast wild type S-II protein was expressed in Escherichia coli and purified as described elsewhere (2Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Google Scholar). Anti-yeast S-II antiserum was prepared at Asahi Techno Glass Corp. (Chiba, Japan) by injecting a New Zealand White rabbit with 0.3 mg of the purified recombinant yeast S-II protein. Anti-yeast S-II antibodies were affinity-purified from the antiserum as previously described (26Olmsted J.B. Methods Enzymol. 1986; 134: 467-472Google Scholar). YPH499 was transformed with pRS416 (Stratagene), designated TU100, and used as the wild type strain for this experiment. Mutant yeast strains Mt1 to Mt9 were also transformed with pRS416. The resulting nine S-II mutant strains, TU100, and an S-II mull mutant strain HKY01 were separately grown in SD (ura−) medium at 30 °C until they reached midlog phase growth, and then cells were collected by centrifugation. A CellLytic Y solution (Sigma) containing 1 mm phenylmethylsulfonyl fluoride, 5 mmbenzamidine, 10 μg/ml leupeptin, and 2 μg/ml pepstatin A was used to lyse each strain separately. Fifty micrograms of protein from the crude lysate was loaded in each lane of a SDS-polyacrylamide gel and then blotted to a Hybond-P membrane (Amersham Biosciences). The blot was incubated with affinity-purified anti-yeast S-II antibodies at 4 °C for 16 h and then with anti-rabbit IgG (donkey) conjugated to horseradish peroxidase (Amersham Biosciences) at 20 °C for 1 h. ECL Plus (Amersham Biosciences) was used for detection. The Kaleidoscope Prestained Standard protein ladder (Bio-Rad) was used to estimate molecular weights. The same yeast strains used in the Western blot analysis were separately incubated in SD (ura−) medium at 30 °C until they reached midlog phase growth, and then 0.18 volume of formaldehyde and 0.13 volume of 1 mpotassium acetate buffer (pH 6.5) were added to the medium. Each culture was incubated at 23 °C for 1 h. Cells were then collected by centrifugation and resuspended in 0.1 mpotassium acetate buffer (pH 6.5) containing 5% formaldehyde and incubated at 23 °C for 1 h. The cells were then collected by centrifugation and washed once with SHA buffer (1 msorbitol, 0.1 m HEPES-Na, pH 7.2, and 5 mmNaN3) and resuspended in SHA. Cells were then treated with Zymolyase 100T (Seikagaku Corp., Tokyo, Japan) and transferred onto ADCELL slides (Erie Scientific Co.; Portsmouth, NH). Cells were fixed in methanol at −20 °C for 6 min and then in acetone at −20 °C for 30 s. Fixed cells were incubated with affinity-purified anti-yeast S-II antibodies or preimmune rabbit IgG followed by incubation with anti-rabbit IgG (goat) conjugated to the Alexa-488 fluorescent dye (Molecular Probes, Inc., Eugene, OR) for detection. The nuclei were stained with 4′,6-diamidino-2-phenylindole. The same yeast strains used in the Western blot analysis were separately incubated in SD (ura−) medium at 30 °C for overnight and then diluted with fresh medium toA 600 = 0.0425. The diluted cultures were incubated until the A 600 reached 0.17 and then diluted 10-, 100-, 1000-, or 10000-fold, and 7 μl of each diluted culture was spotted onto SD (ura−) plates containing 75 μg/ml 6-AU, 25 μg/ml MPA, or no drug. The plates were incubated at 30 °C for 4 days. The same yeast strains used in the Western blot analysis were separately incubated in SD (ura−) medium at 30 °C until midlog phase, and then 6-AU to give 75 μg/ml was added. After 0, 0.5, or 2 h, cells were collected by centrifugation, and a hot phenol method (27Kohrer K. Domdey H. Methods Enzymol. 1991; 194: 398-405Google Scholar) was used to extract total RNA. Fifteen micrograms of total RNA was loaded onto each lane of an agarose gel containing formaldehyde for Northern blot analysis. The probes were the PCR products amplified from yeast genomic DNA with 5′-GTGGTATGTTGGCCGGTACTACCG-3′ and 5′-TCAGTTATGTAAACGCTTTTCGTA-3′ as primers for IMD2 (22Shaw R.J. Reines D. Mol. Cell. Biol. 2000; 20: 7427-7437Google Scholar) and 5′-TGATAACGGTTCTGGTATGTG-3′ and 5′-TAGTCAGTCAAATCTCTACCG-3′ as primers for actin. The AlkPhos Direct Labeling System with CDP-Star (Amersham Biosciences) was used to label the probes with alkaline phosphatase, and the probes were hybridized with the blots at 55 °C for 16 h. The blots were exposed to x-ray films, and the developed films were scanned with a GS-700 calibrated densitometer (Bio-Rad) to quantify the amount of transcript. The signal of the actin mRNA in each sample was used to normalize the band intensity, and the IMD2 signal of the TU100 following a 2-h induction by 6-AU was used as a relative measurement unit. TU100 was transformed with pRS413 (Stratagene) to create TU101. HKY02 was transformed with pRS416 to create TU102. TU101 was used as the wild type strain and TU102 was used as dst1Δin this experiment. Yeast strainsspt4Δand Mt1spt4Δ to Mt9spt4Δ were transformed with pRS413. These strains and dst1Δspt4Δwere separately incubated in SC (ura− his−) medium (0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose, and 0.06% (w/v) complete supplement mixture (−his, −leu, −ura, −trp) from Qbiogene Inc., 0.1 mg/ml each leucine, tryptophan, and lysine, and 0.04 mg/ml adenine sulfate) at 30 °C for 16 h and then diluted toA 600 = 0.085 with YPDA medium (1% (w/v) yeast extract, 2% (w/v) polypeptone, 2% (w/v) glucose, and 0.04 mg/ml adenine sulfate). The diluted cultures were incubated until theA 600 reached 0.17 and then diluted 10-, 100-, 1000-, and 10,000-fold; 7 μl of each culture was then spotted onto YPDA plates. Plates were then incubated at either 30 or 37 °C (restrictive temperature for dst1Δspt4Δ double null mutant) for 5 days. DNA manipulation was performed as described elsewhere (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The method of Bradford (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) was used to determine protein concentration; bovine serum albumin was used as a standard. The S. cerevisiae S-II protein consists of three domains: domains I, II, and III. Domains II and III are joined by a short linker region (30Olmsted V.K. Awrey D.E. Koth C. Shan X. Morin P.E. Kazanis S. Edwards A.M. Arrowsmith C.H. J. Biol. Chem. 1998; 273: 22589-22594Google Scholar). Awrey et al. (31Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Google Scholar) constructed a variety of the S. cerevisiae S-II mutant proteins that span domains II and III. They then determined the in vitro cleavage and read-through-stimulating activities of these mutant S-II proteins with an assay that used a human histone H3.3 gene fragment, which contains well characterized transcriptional arrest sites, as the template (31Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Google Scholar). To see whether both cleavage and read-through stimulating activities of S-II were responsible for its biologic functions, nine of these mutant S-II proteins, designated Mt1 to Mt9, were selected for further tests. Results from the in vitro assays conducted by Awrey et al. (31Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Google Scholar) show that four of the mutant proteins had strong cleavage stimulation but different read-through-stimulating activities (Mt1, -4, -5, and -7); four others (Mt2, -6, -8, and -9) had moderate cleavage stimulation but different read-through-stimulating activities, and Mt3 had little of either activity (Table II). Mt6 has a mutation in domain II, which binds to RNA polymerase II. Mt1, -2, and -7 have mutations in the linker region, and the other mutant proteins have mutations in domain III, which is essential for transcript cleavage and read-through-stimulating activities as well as 6-AU sensitivity suppression (31Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Google Scholar, 32Nakanishi T. Shimoaraiso M. Kubo T. Natori S. J. Biol. Chem. 1995; 270: 8991-8995Google Scholar). These mutations were introduced into the S-II locus of S. cerevisiae by homologous recombination.Table IISummary of the phenotypes of S-II mutantsIn vitroIn vivoS-IICleavageRead-through6-AU resistanceMPA resistanceIMD2 inductionTemperature resistanceWild type33++++Null mutation00−−−−Mt1 (K242A/Q243A)22++++Mt2 (N252A/N255A/Q257A)11+++±Mt3 (R287Q/E291L)00−−−−Mt4 (F296A)21++++Mt5 (K307A)20++++Mt6 (R200A)12+++±Mt7 (F269A)21++++Mt8 (R287Q/E291N)10−−−−Mt9 (R287Q)11++++In vitro activities of the mutants were determined by Awreyet al. (31Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Google Scholar). 0, inactive or partially active at 500:1 (S-II protein:RNA polymerase II); 1, active at 500:1; 2, active at 100:1; 3, active at 5:1 for in vitro activities. +, resistant or strong induction; ±, sensitive; −, very sensitive or marginal induction for in vivo activities. Open table in a new tab In vitro activities of the mutants were determined by Awreyet al. (31Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Google Scholar). 0, inactive or partially active at 500:1 (S-II protein:RNA polymerase II); 1, active at 500:1; 2, active at 100:1; 3, active at 5:1 for in vitro activities. +, resistant or strong induction; ±, sensitive; −, very sensitive or marginal induction for in vivo activities. First, Western blot analysis was used to determine the amount of mutant S-II proteins expressed, and indirect immunofluorescence was used to determine the cellular localization of the mutant S-II proteins; both methods used anti-yeast S-II specific antibodies. Results of the Western blot analysis show that all S-II mutant strains have expression patterns similar to that of the wild type strain (Fig.1). In addition to a major band of 42 kDa, which was as large as the recombinant S-II, a faint band (35 kDa) was observed in all strains. Since this signal was not detected in the S-II null mutant HKY01 (Fig. 1), it is thought that the smaller band represents a degradation product of S-II. Results of preliminary studies suggest that the smaller form lacks the carboxyl terminus region 2N. Adachi and T. Nakanishi, unpublished results. that is essential for RNA polymerase II stimulation, read-through stimulation in vitro, and 6-AU sensitivity suppression in vivo (32Nakanishi T. Shimoaraiso M. Kubo T. Natori S. J. Biol. Chem. 1995; 270: 8991-8995Google Scholar). The band intensity of the Mt4 S-II protein is reproducibly weaker than the S-II proteins from the other mutant strains (Fig. 1). These data suggest that, compared with the S-II proteins from the other mutant strains, the amount of S-II protein expressed by the Mt4 strain was lower, or the antigenicity of Mt4 S-II protein is weaker. However, it should be noted that results presented below indicate that the amount of Mt4 S-II protein expressed was apparently sufficient to perform its biologic functions (Table II). The results of indirect immunofluorescence experiments to locate S-II are shown in Fig. 2. As shown in Fig.2 A, anti-S-II staining of the wild type strain TU100 showed strong fluorescence that overlapped with nuclear staining, as previously reported (33Albertini M. Pemberton L.F. Rosenblum J.S. Blobel G. J. Cell Biol. 1998; 143: 1447-1455Google Scholar). In contrast, no fluorescence was observed in S-II null mutant strain HKY01 stained with anti-S-II (Fig.2 B) or in wild type (Fig. 2 B) and Mt1 to Mt9 (data not shown) cells stained with the same concentration (1.7 μg/ml) of preimmune rabbit IgG. The staining patterns of Mt1 to Mt9 with anti-S-II were indistinguishable from that of the wild type (Fig.2 C) and overlapped with nuclear staining (data not shown). These results indicate that all of the mutant S-II proteins are localized in the nucleus as is the wild type S-II protein. The S. cerevisiae S-II null mutant is known to be sensitive to 6-AU (2Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Google Scholar) and MPA (20Exinger F. Lacroute F. Curr. Genet. 1992; 22: 9-11Google Scholar). To investigate the biologic effects of S-II mutant proteins, the sensitivity of each S-II mutant strain to 6-AU and MPA was tested. Fig. 3 shows that mutant strains Mt1, -2, -4, -5, -6, -7, and -9 are as resistant to 6-AU as the wild type is; in contrast, Mt3 and Mt8 are as sensitive to 6-AU as is the S-II null mutant. Interestingly, despite its inability to promote read-through in vitro (Table II), Mt5 is as resistant to 6-AU as is the wild type. The sensitivities of mutant strains Mt1 to Mt9 to 25 μg/ml MPA correlate well with the sensitivities to 6-AU (Fig. 3). Additionally, introduction of a centromeric plasmid that contained the S. cerevisiae S-II gene complemented the drug sensitivities of Mt3 and Mt8 (data not shown). These results suggest that the read-through-stimulating activity of S-II is not needed for the suppression of 6-AU and MPA sensitivity. TheIMD2 gene encodes IMP dehydrogenase, a key enzyme in thede novo synthesis of guanine nucleotides (34Kohler G.A. White T.C. Agabian N. J. Bacteriol. 1997; 179: 2331-2338Google Scholar). The activity of IMP dehydrogenase is inhibited by MPA and by 6-azauridine monophosphate, an active metabolite of 6-AU (20Exinger F. Lacroute F. Curr. Genet. 1992; 22: 9-11Google Scholar).IMD2 gene transcription can be induced by the addition of 6-AU or MPA to the culture medium (22Shaw R.J. Reines D. Mol. Cell. Biol. 2000; 20: 7427-7437Google Scholar), and this induction depends on S-II. This induction is thought to be important for the development of 6-AU and MPA resistance. Therefore, the ability of 6-AU to induceIMD2 gene transcription in the S-II mutant strains was tested. The wild type TU100 strain, the S-II null mutant HKY01 strain, and the mutant strains Mt1 to Mt9 transformed with pRS416 were incubated in a synthetic medium in the presence or absence of 75 μg/ml 6-AU for up to 2 h, and Northern blot analysis was used to determine the amount of IMD2 mRNA produced by each strain. As shown in Fig. 4, mutant strains Mt1, -2, -4, -5, -6, -7, and -9 induced IMD2 gene expression as well as the wild type strain did, whereas mutant strains Mt3 and Mt8 were as transcriptionally inactive as the S-II null mutant. These data illustrate that the biologic ability to induce theIMD2 gene transcription correlates positively with thein vitro cleavage stimulation activity exhibited by the S-II mutant proteins. SPT4 gene encodes a subunit of the transcription elongation factor DSIF (DRBsensitivity-inducing factor) (35Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Google Scholar). A null mutant of the DST1 gene, which encodes S. cerevisiae S-II, shows temperature sensitivity when combined with the spt4 null mutation, whereas the spt4 null mutant is not temperature-sensitive (17Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Google Scholar). To see whether mutant S-II proteins can suppress the temperature-sensitive phenotype of thedst1Δspt4Δ double null mutant strain, S-II mutant strains carrying the spt4 null mutation were constructed and tested for growth at 37 °C. The results are shown in Fig. 5. Thedst1Δspt4Δ double null mutant strain showed clear temperature sensitivity. Mt1spt4Δ, Mt4spt4Δ, Mt5spt4Δ, Mt7spt4Δ, and Mt9spt4Δ grew as well as spt4Δ at 37 °C, indicating that the Mt1, -4, -5, -7, and -9 S-II mutant proteins suppress temperature sensitivity as well as does the wild type S-II protein. However, the read-through activities of these five S-II mutant proteins vary and do not correlate with their suppression activities. Mt2spt4Δ and Mt6spt4Δ showed moderate temperature sensitivity, indicating that the suppression activities of Mt2 and Mt6 proteins are weaker than that of the wild type S-II protein. Mt3spt4Δ and Mt8spt4Δ were temperature-sensitive, showing that the Mt3 and Mt8 proteins lack suppression activity. These results indicate that the biologic ability to suppress temperature-restricted growth correlates positively with the in vitro cleavage stimulation activities of the S-II mutant proteins. At a transcriptional arrest site in a gene, S-II stimulates the nuclease activity of RNA polymerase II into cleaving several bases from the nascent transcript and causes the realignment of the 3′ end of the nascent transcript with the active site of the polymerase, and the transcription elongation complex tries to read through the arrest site again (11Christie K.R. Awrey D.E. Edwards A.M. Kane C.M. J. Biol. Chem. 1994; 269: 936-943Google Scholar, 12Izban M.G. Luse D.S. Genes Dev. 1992; 6: 1342-1356Google Scholar, 13Reines D. J. Biol. Chem. 1992; 267: 3795-3800Google Scholar, 14Reines D. Ghanouni P. Gu W. Mote Jr., J. Powell W. Cell Mol. Biol. Res. 1993; 39: 331-338Google Scholar). Results from previous studies have shown that this cleavage stimulation activity of S-II is necessary, but not sufficient, to promote read-through in vitro (15Cipres-Palacin G. Kane C.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8087-8091Google Scholar). To clarify this observation, S. cerevisiae S-II mutant strains were used to investigate the roles of the cleavage and read-through stimulation activities of S-II in vivo. A 6-AU sensitivity assay (Fig.3), RNA analysis for IMD2 induction by 6-AU (Fig. 4), and rescue from temperature-dependent growth inhibition of separate strains carrying one of the S-II mutations and anspt4 null mutation (Fig. 5) were used to assess S-II function. The results show that S-II mutant proteins that were active in both cleavage stimulation and read-through in vitro also suppressed 6-AU sensitivity, induced IMD2 gene expression, and suppressed dst1Δspt4Δ temperature-dependent growth inhibition. In contrast, those S-II mutant proteins inactive in vitro did not exhibit any of these biologic functions. Surprisingly, S-II mutant protein Mt5, which has strong cleavage stimulation activity but almost no read-through activity in vitro, performed all biologic functions. Overall, the cleavage-stimulating activities, but not read-through stimulating activities of each mutant S-II protein, corresponded well with their biologic activities. The Mt2, Mt3, Mt6, and Mt8 S-II mutant proteins performed biologic functions not as well as wild type S-II protein (Table II). But the amounts of Mt2, -3, -6, and -8 proteins expressed were as great as the amount of wild type S-II (Fig. 1), and all mutant proteins were localized in the nucleus as the wild type protein was (Fig. 2). Thus, the low biologic activities of Mt2, -3, -6, and -8 S-II mutant proteins were not caused by insufficient protein expression or by cellular mislocalization. These results, summarized in Table II, strongly suggest that the cleavage stimulation activity of S-II alone is responsible for its biologic functions, whereas the read-through stimulation activity of S-II is dispensable. Previously, S-II has been regarded as a transcriptional read-through factor, and its read-through activity has been assumed to be important for its biologic functions; however, the results of the present study do not support this notion. It is possible, however, that the S-II mutant proteins that are inactive in an in vitroread-through assay have sufficient read-through activity in the cell. The results of the present study also imply the presence of some as yet unidentified transcriptional read-through factors. Since the mutant S-II protein Mt5, which lacks read-through activity in vitro, exhibited full biologic activity, it is possible that once the nascent transcript is cleaved by RNA polymerase II, other read-through factors might promote read-through in place of a read-through-incompetent S-II protein in Mt5 strain. Awrey et al. (31Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Google Scholar) suggested that a conformational change in the ternary elongation complex (RNA polymerase II-DNA template-nascent transcript) is required for read-through after the cleavage of the nascent transcript. It has been suggested that S-II and Rpb9 influence the conformational change necessary for read-through (31Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Google Scholar). Thus, Rpb9 is a good candidate for a read-through factor other than S-II. Mutant yeast strains sensitive to 6-AU that bear the Mt5 mutation may be good tools for identifying this putative read-through factor. Another possible approach would be to use an in vitro read-through assay system containing the S-II Mt5 protein to isolate read-through-promoting factors biochemically. These studies, which are currently under way, are the next logical steps in understanding the regulation of transcription elongation by RNA polymerase II in eukaryotic cells. We thank Dr. T. Ito for HKY01 and HKY02, critical reading of the manuscript, and helpful discussions; Dr. M. Nakanishi-Matsui for critical reading of the manuscript and helpful discussions; and Steven E. Johnson for editing the manuscript.