RNA Polymerase II Subunit Rpb9 Regulates Transcription Elongation in Vivo

RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro andin vivo and is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show thatRPB9 has a synthetic phenotype with the TFIIS gene. Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Δrpb9cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of Δrpb9 cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of Δrpb9 cells revealed only a few changes, predominantly in genes related to metabolism. RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro andin vivo and is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show thatRPB9 has a synthetic phenotype with the TFIIS gene. Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Δrpb9cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of Δrpb9 cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of Δrpb9 cells revealed only a few changes, predominantly in genes related to metabolism. RNA polymerase II lacking the Rpb9 subunit RNA polymerase II comprises 12 subunits in yeast (1Sawadogo M. Sentenac A. Annu. Rev. Biochem. 1990; 59: 711-754Crossref PubMed Scopus (310) Google Scholar). Four of the subunits, Rpb1, Rpb2, Rpb3, and Rpb11, form a catalytic core that is homologous in structure and function to the prokaryotic core RNA polymerase (2Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 3Cramer P. Bushnell D.A. Fu J. Gnatt A.L. Maier-Davis B. Thompson N.E. Burgess R.R. Edwards A.M. David P.R. Kornberg R.D. Science. 2000; 288: 640-649Crossref PubMed Scopus (472) Google Scholar). The other eight eukaryotic subunits are less well characterized. Five of these subunits, Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are found in all three eukaryotic RNA polymerases (4Carles C. Treich I. Bouet F. Riva M. Sentenac A. J. Biol. Chem. 1991; 266: 24092-24096Abstract Full Text PDF PubMed Google Scholar, 5Woychik N.A. Young R.A. J. Biol. Chem. 1990; 265: 17816-17819Abstract Full Text PDF PubMed Google Scholar, 6Woychik N.A. Liao S.-M. Kolodziej P.A. Young R.A. Genes Dev. 1990; 4: 313-323Crossref PubMed Scopus (138) Google Scholar). The other three, Rpb4, Rpb7, and Rpb9, are unique to RNA polymerase II, although both Rpb7 and Rpb9 have sequence homologues in RNA polymerases I and III (7Nogi Y. Yano R. Dodd J. Carles C. Nomura M. Mol. Cell. Biol. 1993; 13: 114-122Crossref PubMed Scopus (97) Google Scholar). The gene for Rpb9 is not essential for yeast cell viability, but is essential in Drosophila(8Harrison D.A. Mortin M.A. Corces V.G. Mol. Cell. Biol. 1992; 12: 928-935Crossref PubMed Scopus (43) Google Scholar). Rpb9 has roles both in transcription initiation and in transcription elongation. In the initiation reaction, Rpb9 modulates the selection of the transcription start site. In cells lacking Rpb9 and in reconstituted transcription reactions lacking Rpb9, the population of start sites is shifted upstream at a variety of promoters (9Furter-Graves E.M. Hall B.D. Furter R. Nucleic Acids Res. 1994; 22: 4932-4936Crossref PubMed Scopus (39) Google Scholar, 10Hull M.W. McKune K. Woychik N.A. Genes Dev. 1995; 9: 481-490Crossref PubMed Scopus (88) Google Scholar, 11Sun Z.W. Tessmer A. Hampsey M. Nucleic Acids Res. 1996; 24: 2560-2566Crossref PubMed Scopus (50) Google Scholar). In the elongation reaction, Rpb9 is required, along with TFIIS, to effect transcription through blocks to elongation encoded by the DNA template (12Awrey D.E. Weilbaecher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). A role in the modulation of initiation and elongation is consistent with the localization of Rpb9 in the three-dimensional structure of yeast RNA polymerase II. Rpb9 is located at the tip of the so-called “jaws” of the enzyme, which is thought to function by clamping the DNA downstream of the active site (3Cramer P. Bushnell D.A. Fu J. Gnatt A.L. Maier-Davis B. Thompson N.E. Burgess R.R. Edwards A.M. David P.R. Kornberg R.D. Science. 2000; 288: 640-649Crossref PubMed Scopus (472) Google Scholar, 13Poglitsch C.L. Meredith G.D. Gnatt A.L. Jensen G.J. Chang W.H. Fu J. Kornberg R.D. Cell. 1999; 98: 791-798Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Fu J. Gnatt A.L. Bushnell D.A. Jensen G.J. Thompson N.E. Burgess R.R. David P.R. Kornberg R.D. Cell. 1999; 98: 799-810Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The Rpb9 homologue in RNA polymerase III, C11, also has been implicated in regulating RNA chain elongation (15Chedin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (152) Google Scholar). Rpb9 comprises two zinc ribbon domains joined by a 30-amino acid linker. The C-terminal zinc ribbon is a sequence homologue of the zinc ribbon in the transcription elongation factor TFIIS (16Wang B. Jones D.N. Kaine B.P. Weiss M.A. Structure. 1998; 6: 555-569Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 17Qian X. Jeon C. Yoon H. Agarwal K. Weiss M.A. Nature. 1993; 365: 277-279Crossref PubMed Scopus (111) Google Scholar). The roles of each domain of Rpb9 in transcription elongation were determined by assaying a series of alanine-scanning mutants of Rpb9 in in vitro reactions (18Hemming S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Alanine substitutions in the C-terminal zinc ribbon domain of Rpb9, like amino acid substitutions in the homologous part of TFIIS, completely eliminated elongation activity. Mutating the first zinc ribbon had no effect on elongation activity, although deleting this domain entirely abrogated activity. The linker region mediated the interaction of Rpb9 with the rest of the RNA polymerase. In this study, we used this series of mutations to probe the cellular role of Rpb9 in both initiation and elongation. YF2221 (MAT a ura3-52 his3-11,15 leu2-3,112 ade2-1 can1-100 ssd1-d2 trp1::hisG-URA3-hisG) is the parent strain. YF2230 (MAT a ura3-52 his3-11,15 leu2-3,112 ade2-1 can1-100 ssd1-d2 trp1::hisG-URA3-hisG rpb9::HIS3) is a derivative of YF2221 deleted for RPB9. YF2222 (MAT a ura3-52 his3-11,15 leu2-3,112 ade2-1 can1-100 ssd1-d2 trp1-1 ppr2::hisG-URA3-hisG) is deleted for the TFIIS gene, and YF2234 (MAT a ura3-52 his3-11,15 leu2-3,112 ade2-1 can1-100 ssd1-d2 trp1-1 ppr2::hisG-URA3-hisG rpb9::HIS3) lacks bothRPB9 and the TFIIS gene. The yeast expression plasmid pRS314RPB9 containing theRPB9 open reading frame plus ∼500 base pairs upstream and 2200 base pairs downstream was obtained from Dr. Rolf Furter (9Furter-Graves E.M. Hall B.D. Furter R. Nucleic Acids Res. 1994; 22: 4932-4936Crossref PubMed Scopus (39) Google Scholar). This plasmid was adapted by inserting a BamHI restriction site immediately upstream of the start codon and an EcoRI restriction site immediately downstream of the stop codon, creating the plasmid pRS314RPB9BE. These sites were inserted using the QuikChange protocol and PfuI DNA polymerase (Stratagene). Incorporation of these restriction sites allowed for the insertion of each of the previously constructed rpb9 mutants into pRS314RPB9BE. The resulting plasmids, containing each of therpb9 mutant alleles under control of the endogenousRPB9 promoter, were transformed into yeast to determine their effects on growth and the use of initiation start sites. The Δrpb9 yeast strain grows slowly at 30 °C, is extremely sensitive to high- and low-temperature extremes, and is sensitive to the drug 6-azauracil. Expression of wild-typeRPB9 corrects these defects. Haploid Δrpb9cells were transformed with the RPB9 yeast expression plasmids to test each mutant for the ability to complement theΔrpb9 growth phenotypes. To test for complementation of cold and temperature sensitivity, the cells were grown on solid synthetic complete yeast medium lacking tryptophan. Suspensions containing ∼10,000, 2000, 400, and 80 cells were spotted onto solid medium and grown at 12, 30, or 37 °C for 2–6 days. Cells were grown on solid synthetic complete yeast medium lacking tryptophan and uracil and containing 50 μg/ml 6-azauracil to measure ability to correct sensitivity to 6-azauracil. Suspensions containing ∼10,000, 2000, 400, and 80 cells were spotted onto solid medium and grown at 30 °C for 3–8 days. Each mutant construct was compared with wild-typeRPB9 with respect to ability to restore growth characteristics. Primer extension assays were performed to identify the transcription start sites in the mutant yeast strains. Yeast strains YF2221, YF2222, YF2230, and YF2234 were grown in yeast extract peptone liquid medium with 2% glucose. YF2230 cells transformed with each of the pRS314RPB9BE constructs was grown in liquid complete synthetic medium lacking tryptophan. All cultures were grown at 30 °C to A 600 nm = 0.2 to 1.0. Cells (5 × 107) were harvested, and total RNA was isolated using the RNeasy protocol (QIAGEN Inc.). The primer used for these experiments, 5′-AGAAGATAACACCTTTTTGAG-3′ (Dalton Chemicals), is complementary to nucleotides +37 to +17 in the ADH1 gene. The primer was radiolabeled at the 5′-end by phosphorylating with polynucleotide kinase (New England Biolabs Inc.) and [γ-32P]ATP. For each primer extension reaction, 15 μg of total RNA from the appropriate yeast strain was annealed with 0.4 pmol of the 5′-radiolabeled primer for 45 min at 52 °C. Reverse transcription from the annealed primer was done with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) according to manufacturer's instructions. The reverse transcripts were collected by ethanol precipitation and resolved on a Tris borate/EDTA, 8.3 m urea, and 6% polyacrylamide gel and visualized by phosphorimaging. Yeast cells were grown in yeast extract peptone 2% glucose medium at 30 °C with constant agitation and aeration to A 600 nm = 0.4–0.6. Cells were washed once with diethyl pyrocarbonate-treated water and in containing and RNA was isolated using a R. K. in and using a RNeasy by the RNA were determined by the at RNA were for each mutant strain. For each DNA 50 μg of total RNA was using of II (Life Technologies, Inc.). The reverse transcription was with an primer and performed in the of of of 50 and or of 50 20 of was also to the The the was at °C for min and at °C for the reverse transcription was and the was at °C for The reverse transcription was with of and the RNA template was by the of of and at °C for 20 The was by the of m acid of and the was by the of of and on for 30 with the was in μg of diethyl pyrocarbonate-treated For each DNA of and of were to of of yeast and of DNA were also to the and the was at °C for The was under a to a yeast The were at 37 °C in a for the were washed with and × 15 min at 50 with × min at and by A total of eight were for each mutant strain. The were on a and the obtained were using implicated Rpb9 in transcription elongation in vitro (12Awrey D.E. Weilbaecher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In cells lacking Rpb9, a of the RNA polymerase II transcription at promoters at upstream DNA This by the of wild-type but not a mutant in the N-terminal zinc ribbon revealed a role for Rpb9 in transcription elongation in vitro (12Awrey D.E. Weilbaecher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In (12Awrey D.E. Weilbaecher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google the mutant was to have the elongation the wild-type RNA polymerase II, but less at DNA a sequence from the that of the transcription The of Rpb9 to restored in the form elongation at the site. wild-type these were unable to by the of the elongation factor In these revealed a role for Rpb9 in transcription elongation. The of Rpb9 that to the elongation were determined using a set of Rpb9 deletion and alanine-scanning mutants (18Hemming S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These that the C-terminal zinc ribbon domain was for was the linker region the two zinc Rpb9. The linker region was to for the of Rpb9 to We were unable to show that the N-terminal zinc which is for start site selection M.W. McKune K. Woychik N.A. Genes Dev. 1995; 9: 481-490Crossref PubMed Scopus (88) Google a role in elongation. TFIIS and Rpb9 are linked and are related in We were is also a interaction and TFIIS gene TFIIS gene is also Yeast cells lacking the gene for Rpb9 are sensitive to both and and slowly wild-type strains at the growth temperature M.W. McKune K. Woychik N.A. Genes Dev. 1995; 9: 481-490Crossref PubMed Scopus (88) Google N.A. Young R.A. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). These phenotypes were also in and normal growth was restored by from a plasmid under the control of Yeast strains lacking the TFIIS gene are sensitive to the drug 6-azauracil. This phenotype is thought to a in transcription elongation F. F. 1992; 22: PubMed Scopus Google Scholar). Rpb9 is for the interaction between RNA polymerase II and TFIIS, we the Δrpb9 strain for sensitivity to 6-azauracil. strain slowly on medium containing 6-azauracil the parent strain the with the RPB9 gene on a plasmid complemented the 6-azauracil sensitivity The deletion strain was and phenotype was to the interaction between the TFIIS gene and RPB9. In with the the mutant a phenotype of the gene These are consistent with a interaction between TFIIS and Rpb9 in vivo. The and Δrpb9 strains were each transformed with high-copy plasmids the wild-type TFIIS gene and These and control strains were for growth on containing and μg/ml 6-azauracil. The TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Δrpb9 In on a high-copy plasmid not the 6-azauracil sensitivity of These that the elongation by the of Rpb9 restored partially by the cellular of We that the effects on cell growth by in part from defects in transcription elongation. Rpb9 was to have a role in regulating the of the transcription start sites (9Furter-Graves E.M. Hall B.D. Furter R. Nucleic Acids Res. 1994; 22: 4932-4936Crossref PubMed Scopus (39) Google Scholar, 10Hull M.W. McKune K. Woychik N.A. Genes Dev. 1995; 9: 481-490Crossref PubMed Scopus (88) Google Scholar, 11Sun Z.W. Tessmer A. Hampsey M. Nucleic Acids Res. 1996; 24: 2560-2566Crossref PubMed Scopus (50) Google Rpb9 was implicated in transcription elongation (12Awrey D.E. Weilbaecher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Here, we the of the set of rpb9 mutants in vivo to into the role of Rpb9. The 20 mutants were for their ability to restore normal growth to and yeast To each strain was transformed with the plasmid each of the rpb9 mutant and the phenotypes were All of the 20 alanine-scanning Rpb9 mutants restored normal growth to Δrpb9 These mutants into two that had no effect on of the other Rpb9 site selection and not and that or the other first and mutants had no effect on growth initiation in or elongation in vitro and are not in and These mutations are located in the N-terminal zinc ribbon and the linker of in vivo of rpb9 mutant in previously completely normal growth at 30 °C on synthetic complete solid medium lacking partially normal growth at 30 °C on synthetic complete solid medium lacking not restore normal growth at 30 °C on synthetic complete solid medium lacking completely normal growth at 30 °C on synthetic complete solid medium lacking partially normal growth at 30 °C on synthetic complete solid medium lacking not restore normal growth at 30 °C on synthetic complete solid medium lacking previously (18Hemming S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). in a rpb9 alleles with or or N-terminal in of the two zinc domains had cell growth phenotypes first and A C-terminal which the N-terminal domain and part of the linker region but lacks the zinc was unable to restore normal cell growth to Δrpb9 deletion mutants in the first zinc region and also were unable to complement We that Rpb9 both for normal For all rpb9 mutants the three and 6-azauracil sensitivity, were This suggests that the of Rpb9 is the primary that all three phenotypes and that are not effects of the gene rpb9 cells for transcription start sites on a variety of promoters (9Furter-Graves E.M. Hall B.D. Furter R. Nucleic Acids Res. 1994; 22: 4932-4936Crossref PubMed Scopus (39) Google Scholar, 10Hull M.W. McKune K. Woychik N.A. Genes Dev. 1995; 9: 481-490Crossref PubMed Scopus (88) Google Scholar, 11Sun Z.W. Tessmer A. Hampsey M. Nucleic Acids Res. 1996; 24: 2560-2566Crossref PubMed Scopus (50) Google Scholar). In an upstream of the 5′-end of the is In this study, the ADH1 gene, which a in the transcription start site between the wild-type parent and the Δrpb9 strains M.W. McKune K. Woychik N.A. Genes Dev. 1995; 9: 481-490Crossref PubMed Scopus (88) Google was used to the effect of the rpb9 alleles on the use of initiation sites. A strain and a strain were also to determine the effect of TFIIS on initiation start site Primer extension was performed on RNA isolated from the different yeast strains using a primer the 5′-end of the ADH1 gene. The pattern of initiation sites in the wild-type strain was compared with in the strains. The RNA for this primer extension was from these strains were grown in a The of initiation sites in the wild-type and Δrpb9 strains are the reverse transcripts from the Δrpb9 strain are and an in the of that start upstream of The deletion of the TFIIS gene to have no on start site in with the deletion of RPB9. strain had a transcription initiation to that of the wild-type and the for strain was to that of the Δrpb9 strain. The synthetic phenotype that in the strain not to from a in transcription The of rpb9 alleles restored the normal pattern of initiation sites in yeast cells and was an rpb9 mutant containing only the This which at wild-type M. was to complement the start site but not the other The of this mutant to the role of Rpb9 in transcription initiation from the roles in transcription elongation and cell The phenotypes of the rpb9 mutants are with an role for Rpb9 in transcription Rpb9 structure to function of the for transcription elongation (18Hemming S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). the C-terminal zinc ribbon was for RNA in transcription and for in with TFIIS, and the linker region mediated the interaction of Rpb9 with RNA polymerase II. for in yeast rpb9 mutant that a growth phenotype was for transcription elongation. all of the alanine-scanning mutants that elongation had normal growth and were to restore the normal pattern of initiation start sites We that the in vivo complementation assays are less sensitive of Rpb9 function the in The phenotypes of the strains and their interaction that the have a effect on elongation. To determine a set of genes is by the two transcription we compared the of gene expression in strains using yeast DNA Disruption of the TFIIS gene or RPB9 in cells grown in medium had effect on gene expression not and C. M. the effect was transcription of only of yeast genes was compared with the of these genes to For the expression of a set of was in the Δrpb9 this suggests form of the of gene expression not the to glucose Science. 1997; PubMed Scopus Google Scholar). to determine the is a effect on gene or a of transcription in Δrpb9 cells grown in medium compared with the II I kinase kinase acid and of amino acid genes amino acid amino acid amino acid II in homologue of homologue to and RNA of the RNA of in are gene sequence open reading in a are gene sequence open reading In cells lacking both TFIIS and Rpb9, the were but the an of the RPB9 In the transcription with the growth of the were in the in the deleted rpb9 We were unable to to elongation defects. Rpb9 a role in the sites of transcription Cells lacking Rpb9 transcription at upstream sites on We that a derivative of Rpb9 containing only the and linker domains of Rpb9 was to correct this In the domain by deleting the of the region between the two pairs of or the first M.W. McKune K. Woychik N.A. Genes Dev. 1995; 9: 481-490Crossref PubMed Scopus (88) Google ability to the wild-type initiation sites. these two that the domain the selection of transcription initiation sites. In the structure of yeast RNA polymerase II (3Cramer P. Bushnell D.A. Fu J. Gnatt A.L. Maier-Davis B. Thompson N.E. Burgess R.R. Edwards A.M. David P.R. Kornberg R.D. Science. 2000; 288: 640-649Crossref PubMed Scopus (472) Google Rpb9 is the subunit and is to the DNA downstream of the active site. The and domains are on of a that the DNA from the of the with respect to the The domain to to the DNA template the The of the domain in transcription start sites is consistent with in the RNA polymerase. In to of transcription initiation strains also growth at an sensitivity to and F. F. 1992; 22: PubMed Scopus Google and a for the drug 6-azauracil. of the of this is that the temperature and drug phenotypes from the in start site mutations in the domain were unable to correct the in start site the growth defects. in Δrpb9 we that the in start site selection not to the for the in The domain has a homologue in the domain is essential for elongation activity. mutants or deletion mutants in the in the domain not to an role in the transcription start sites. Each of the amino acid mutants the well the which has the restored the wild-type pattern of initiation sites. The of of the domain of the Rpb9 with RNA polymerase to restore elongation to RNA polymerase II. In we that the mutant the domain and part of the linker with (18Hemming S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). this mutant was to restore correct start site selection in vivo. The between in vitro and in vivo is of the mutants that to in vitro restored both start site and growth in vivo. we that the of Rpb9 of the in the RNA polymerase structure (3Cramer P. Bushnell D.A. Fu J. Gnatt A.L. Maier-Davis B. Thompson N.E. Burgess R.R. Edwards A.M. David P.R. Kornberg R.D. Science. 2000; 288: 640-649Crossref PubMed Scopus (472) Google to a of between Rpb9 and of these the that the of these is for the of the domain into RNA polymerase II in the We the of the DNA at the and for with of

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