Mediator as a General Transcription Factor

Abstract

Others have shown that yeast strains bearing a ts mutation in the Srb4 subunit of Mediator cease transcription of all mRNA at the restrictive temperature, in a manner virtually indistinguishable from a strain bearing a ts mutation in the largest subunit of RNA polymerase II. We find that srb4ts Mediator is defective for the stimulation of basal RNA polymerase II transcription at the restrictive temperature in vitro. Taken together, these findings lead to the suggestion that Mediator is required for basal RNA polymerase II transcription in vivo. On this basis, Mediator is identified as a general transcription factor, comparable in importance to RNA polymerase II and other general factors for the initiation of transcription. The possibility that Mediator serves as an anti-inhibitor, opposing the effects of global negative regulators, is largely excluded. Others have shown that yeast strains bearing a ts mutation in the Srb4 subunit of Mediator cease transcription of all mRNA at the restrictive temperature, in a manner virtually indistinguishable from a strain bearing a ts mutation in the largest subunit of RNA polymerase II. We find that srb4ts Mediator is defective for the stimulation of basal RNA polymerase II transcription at the restrictive temperature in vitro. Taken together, these findings lead to the suggestion that Mediator is required for basal RNA polymerase II transcription in vivo. On this basis, Mediator is identified as a general transcription factor, comparable in importance to RNA polymerase II and other general factors for the initiation of transcription. The possibility that Mediator serves as an anti-inhibitor, opposing the effects of global negative regulators, is largely excluded. Mediator was discovered in 1990 in a yeast transcription system (1Kelleher III, R.J. Flanagan P.M. Kornberg R.D. Cell. 1990; 61: 1209-1215Abstract Full Text PDF PubMed Scopus (288) Google Scholar) and shown to support transcriptional activation (2Flanagan P.M. Kelleher III, R.J. Sayre M.H. Tschochner H. Kornberg R.D. Nature. 1991; 350: 436-438Crossref PubMed Scopus (260) Google Scholar). Purification of Mediator to homogeneity (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (886) Google Scholar) led to the finding of two further activities, stimulation of basal transcription and stimulation of TFIIH protein kinase. The occurrence in Mediator of certain subunits, implicated by genetic screens in the negative regulation of transcription, revealed yet another Mediator activity, its function as a co-repressor (4Li Y. Bjorklund S. Jiang Y.W. Kim Y.J. Lane W.S. Stillman D.J. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10864-10868Crossref PubMed Scopus (217) Google Scholar, 5Gustafsson C.M. Myers L.C. Li Y. Redd M.J. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. 1997; 272: 48-50Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 6Myers L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (252) Google Scholar). Co-activators were reported in a human transcription system in 1991 but were believed at the time to be unrelated to yeast Mediator (7Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Abstract Full Text PDF PubMed Scopus (225) Google Scholar). Attention focused instead on TAFs as responsible for the transmission of regulatory information in metazoan systems (8Pugh B.F. Tjian R. Cell. 1990; 61: 1187-1197Abstract Full Text PDF PubMed Scopus (736) Google Scholar). It was only in 1998 that protein complexes in mammalian cells were identified as counterparts of yeast Mediator and shown to support transcriptional activation (9Jiang Y.W. Veschambre P. Erdjument-Bromage H. Tempst P. Conaway J.W. Conaway R.C. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8538-8543Crossref PubMed Scopus (270) Google Scholar, 10Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Naar A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (637) Google Scholar, 11Sun X. Zhang Y. Cho H. Rickert P. Lees E. Lane W. Reinberg D. Mol. Cell. 1998; 2: 213-222Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 12Naar A.M. Beaurang P.A. Zhou S. Abraham S. Solomon W. Tjian R. Nature. 1999; 398: 828-832Crossref PubMed Scopus (373) Google Scholar, 13Gu W. Malik S. Ito M. Yuan C.X. Fondell J.D. Zhang X. Martinez E. Qin J. Roeder R.G. Mol. Cell. 1999; 3: 97-108Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Human Mediator has since been reported to stimulate basal transcription as well (14Mittler G. Kremmer E. Timmers H.T. Meisterernst M. EMBO Rep. 2001; 2: 808-813Crossref PubMed Scopus (103) Google Scholar, 15Baek H.J. Malik S. Qin J. Roeder R.G. Mol. Cell Biol. 2002; 22: 2842-2852Crossref PubMed Scopus (109) Google Scholar). Studies in the human system provided evidence for direct activator-Mediator interaction. Thyroid hormone receptor (16Fondell J.D. Ge H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8329-8333Crossref PubMed Scopus (462) Google Scholar), SREBP (12Naar A.M. Beaurang P.A. Zhou S. Abraham S. Solomon W. Tjian R. Nature. 1999; 398: 828-832Crossref PubMed Scopus (373) Google Scholar), adenoviral E1A (17Boyer T.G. Martin M.E. Lees E. Ricciardi R.P. Berk A.J. Nature. 1999; 399: 276-279Crossref PubMed Scopus (253) Google Scholar), and other transcriptional regulatory proteins were isolated from human cells as tight complexes with Mediator. Activator-Mediator interaction has been reported in the yeast system (18Park J.M. Kim H.S. Han S.J. Hwang M.S. Lee Y.C. Kim Y.J. Mol. Cell. Biol. 2000; 20: 8709-8719Crossref PubMed Scopus (102) Google Scholar, 19Hengartner C.J. Thompson C.M. Zhang J. Chao D.M. Liao S.M. Koleske A.J. Okamura S. Young R.A. Genes Dev. 1995; 9: 897-910Crossref PubMed Scopus (189) Google Scholar, 20Koh S.S. Ansari A.Z. Ptashne M. Young R.A. Mol. Cell. 1998; 1: 895-904Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), but tight complexes have not yet been demonstrated in yeast cell extracts or in derived fractions. Mediator also interacts directly with RNA polymerase II (pol II). 2The abbreviations used are: pol IIpolymerase IIGSTglutathione S-transferaseHAhemagglutininCTDC-terminal domain. A Mediator-pol II complex was initially isolated from yeast (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (886) Google Scholar, 21Thompson C.M. Koleske A.J. Chao D.M. Young R.A. Cell. 1993; 73: 1361-1375Abstract Full Text PDF PubMed Scopus (388) Google Scholar) and has been verified by both structural (22Asturias F.J. Jiang Y.W. Myers L.C. Gustafsson C.M. Kornberg R.D. Science. 1999; 283: 985-987Crossref PubMed Scopus (201) Google Scholar, 23Davis J.A. Takagi Y. Kornberg R.D. Asturias F.A. Mol. Cell. 2002; 10: 409-415Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) and functional analyses. A chain of communication from activator to Mediator to pol II is therefore believed to underlie transcriptional activation (24Kornberg R.D. Trends Biochem. Sci. 2005; 30: 235-239Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 25Chadick J.Z. Asturias F.J. Trends Biochem. Sci. 2005; 30: 264-271Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 26Conaway R.C. Sato S. Tomomori-Sato C. Yao T. Conaway J.W. Trends Biochem. Sci. 2005; 30: 250-255Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 27Kim Y.J. Lis J.T. Trends Biochem. Sci. 2005; 30: 245-249Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 28Kornberg R.D. Trends Biochem. Sci. 2005; 30: 235-239Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 29Malik S. Roeder R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). polymerase II glutathione S-transferase hemagglutinin C-terminal domain. Mediator-pol II interaction may also underlie the stimulation of basal transcription. Genetic studies, however, have lent credence to an alternative mechanism. Temperature-sensitive mutants in two Mediator subunits, Srb4 and Srb6, exhibit a remarkable phenotype at a restrictive temperature, the rapid cessation of transcription of all genes tested (30Thompson C.M. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4587-4590Crossref PubMed Scopus (209) Google Scholar). This finding has been extended by microarray analysis to 5361 yeast genes, all but two of which showed as great an effect of the srb4ts mutant on transcription as that of a mutation in pol II itself (31Holstege F.C. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1596) Google Scholar). A screen for suppressors of the srb4ts mutation identified subunits of the previously described Not and NC2 protein complexes (32Gadbois E.L. Chao D.M. Reese J.C. Green M.R. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3145-3150Crossref PubMed Scopus (54) Google Scholar, 33Lee T.I. Wyrick J.J. Koh S.S. Jennings E.G. Gadbois E.L. Young R.A. Mol. Cell Biol. 1998; 18: 4455-4462Crossref PubMed Scopus (85) Google Scholar). These complexes are believed to be inhibitors of transcription, raising the possibility that Mediator serves as an anti-inhibitor. Mediator might stimulate basal transcription by relieving repression due to global negative regulatory factors. We report here on a study of the srb4ts mutant Mediator in vitro. The results are informative about the mutant phenotype and the role of Mediator in vivo. They provide a test of the anti-inhibitor idea and illuminate the Mediator mechanism, especially in regard to the stimulation of basal transcription. Reversion of srb4ts Mutations and Phenotypic Analysis—The yeast shuttle vector pCT181, carrying the srb4–138 mutant allele, was recovered from yeast strain Z628 (30Thompson C.M. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4587-4590Crossref PubMed Scopus (209) Google Scholar). The shuttle vector pCT127, carrying the wild type SRB4 gene, was recovered from the isogenic yeast strain Z579. The mutations in pCT181 were reverted one at the time by QuikChange (Qiagen), and the sequences were verified by DNA sequencing. Then the shuttle vectors were introduced into yeast strain Z572 by plasmid shuffling. For controls, the wild type vector, pCT127, and the mutant, pCT181 were also introduced into yeast strain Z572. The isolated yeast strains were grown in YPD (2% (w/v) Bacto Peptone, 1% (w/v) yeast extract, 2% (w/v) glucose) overnight. The cells (3 μlof culture diluted to an A600 of 1) were streaked on YPD plates and grown at or of yeast and genes were by and and and and and of the and The of yeast and genes were by and and of the vectors and Mediator and of the proteins and were as described Y. H. A. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (33) Google Scholar) with one instead of with the was from the by of protein with to the and were and cell were by the the was The were by at for and were on a of with For and with of proteins were with was with and and of of the proteins Srb6, and in and were by and revealed by with The and protein were and in Srb4 and proteins were by with the and with was from D. Stillman of was a from S. Bjorklund and were as described L.C. Gustafsson C.M. Bushnell D.A. Lui M. Erdjument-Bromage H. Tempst P. Kornberg R.D. Genes Dev. 1998; 12: 45-54Crossref PubMed Scopus (252) Google and were from C. Gustafsson and were from C. of was a from D. of were a from M. of analysis was as described Y. H. A. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (33) Google Scholar). of vector was by the vector A. D. 2000; PubMed Scopus Google Scholar) as The was by the to by the of the the of the by QuikChange The from the vector G. J. V. P.A. 1991; PubMed Scopus Google Scholar), the gene, was and into the and of G. A. M. M. 1999; PubMed Scopus Google Scholar), the vector The was by a by the DNA sequences into and of the yeast vector of was introduced at the of wild type and mutant genes in and pCT181 by QuikChange and of the were introduced at the of in both wild type and mutant yeast strains by from with the as described A. D. 2000; PubMed Scopus Google Scholar). The were used to yeast strain Z572 yeast strain and were into by plasmid the yeast strains and the G. A. M. M. 1999; PubMed Scopus Google Scholar), was with as and with The were used to yeast strain the yeast strain Purification of and srb4ts strain or was grown in of YPD (w/v) Bacto Peptone, 2% (w/v) yeast extract, (w/v) glucose) to an A600 of cells were in as described Y. H. A. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (33) Google Scholar) in a at for with of A of cells were at and of was The was at for and by in a at for and in a at for The was the A for to a of A with the and to a of was with and as described (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (886) Google Scholar). The from of cells in this were and the for to the of the with the and to a with The was with of and with a from to The Mediator at Mediator were by with a to type or to be to the an of the was on a with and at the was with of the of srb4ts was to at a of or a in a of The was with and by for at temperature in the and The of and was to an of of only one not only in a but also in of subunits from srb4ts Mediator. The was for and was to and C-terminal cell extracts of wild type and srb4–138 strains (30Thompson C.M. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4587-4590Crossref PubMed Scopus (209) Google Scholar) were and used for transcription as described M. P.A. J. J.A. Mol. Cell. Biol. 1991; PubMed Scopus Google Scholar). with proteins was as described (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (886) Google Scholar) with the and were only a The of was instead of was the was For of on an basis, of was to the the of the was with a and The was as described (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (886) Google Scholar). Purification of from the yeast strain was as described Y. H. A. Erdjument-Bromage H. Tempst P. Kornberg R.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (33) Google Scholar) with the was used instead of in the the at for the was at for and on a in A at the was with of and inhibitors and of The was with and by in the at were further on a as described G. A. M. M. 1999; PubMed Scopus Google Scholar) that both and and The was as described and Purification of and were as described The vectors for and were a from G. J. M. M. G. Meisterernst M. EMBO J. 2000; PubMed Scopus Google Scholar). the the was in of by and on a of with The was with of and of and was with For of of both were diluted with on for and on a with The NC2 was by a of to The was as described by srb4ts Cell with the a for Mediator in in cell extracts were from wild type and srb4ts yeast strains as The extracts were in protein in of general transcription factors and Mediator Srb4 all by and in pol II at a temperature of the extracts comparable of transcription from a used the to a a restrictive temperature of transcription by the srb4ts was that by the wild type The of transcription in the mutant was to or and to transcription was to the wild type not was therefore not a of or the to as a transcription of an by srb4ts J.A. S. Genes Dev. 1999; PubMed Scopus Google Scholar) the possibility of of Srb4 protein in the mutant in this may be to of the complex to transcription, in of Srb4 due to of the mutant Mediator in described results were with a of transcription by the mutant was of the or adenoviral or the or activator protein was and was the stimulation of basal transcription by Mediator was the of this with the mutant that Mediator a role in transcription in vivo. It is not only for transcription for transcription at Mediator a in the of of wild type Mediator to srb4ts transcription at of general transcription factors and pol or in at in the extract, was We the of in the srb4ts at was directly to the of Mediator function and not due to an of the mutant Mediator the of another We further the role of Mediator in basal transcription is from the of the other transcription We the of by wild type Mediator to Mediator on the transcription Mediator was to srb4ts at at or the of was to further initiation and only RNA chain The results were of Mediator or transcription activity, at the time as or time transcription at We that Mediator in the initiation of transcription. other the temperature was from to at or transcription the of to srb4ts or in transcription at A temperature and a of transcription comparable with that at of in wild type extract, of a of initiation and not at of transcription. This provided further evidence for a role of Mediator in and demonstrated a of of Mediator in It demonstrated a for Mediator for of transcription. Genetic and of srb4ts Mediator a in as to at and at the srb4ts revealed from wild type which was responsible for the the mutant was reverted to wild type at of the in and in a of the temperature and as the and required for the of these and were introduced and in into wild type to temperature and the for not The for the of the protein at restrictive as to an alternative as interaction with an with of the mutant of Mediator from the srb4ts yeast strain was subunits, were by the We and at a with an on the of a temperature and protein and of the with both and of wild type and srb4ts on the of were Srb4 protein was due to the analysis with Mediator subunits revealed all in the srb4ts Mediator A in the at the for and the of these subunits as Mediator were for in transcription with and pol II. type and srb4ts basal transcription to about the at A and in a system is of a role of Mediator in basal transcription, from The of wild type Mediator was about at that of srb4ts Mediator by about at the temperature This of Mediator further evidence that the srb4ts phenotype is direct and not a effect of of another transcription of basal transcription by srb4ts Mediator was in the system in of in the with of in the The mutant protein may be in the extract, due to in the by or other Mediator may in the extract, not required in the as a role in H. C. Zhang Hwang M.J. C. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). The srb4ts Mediator transcription at at about the of wild type Mediator The may the of subunits to not but other subunits may have been as the effect of the srb4ts mutation on the stimulation of TFIIH TFIIH is responsible for of the of pol required for the initiation of transcription P. J.M. Trends Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar). due to the srb4ts mutation for the mutant The srb4ts however, of comparable with of wild type Mediator at both and Mediator Not the of subunits of the Not complex and two subunits of NC2 were identified in a screen for suppressors of the phenotype of the srb4ts mutation (32Gadbois E.L. Chao D.M. Reese J.C. Green M.R. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3145-3150Crossref PubMed Scopus (54) Google Scholar, 33Lee T.I. Wyrick J.J. Koh S.S. Jennings E.G. Gadbois E.L. Young R.A. Mol. Cell Biol. 1998; 18: 4455-4462Crossref PubMed Scopus (85) Google Scholar). the Not complex and NC2 have been as general negative of transcription in vivo. We to opposing effects of these negative and Mediator in vitro. We the Not complex from yeast by with the of a The subunits were identified by and and with the of which was in a The complex to transcription in or in the system and was and its effect was with that of interaction the two complexes in vitro. NC2 was in and by The protein with transcription in the in with results of (32Gadbois E.L. Chao D.M. Reese J.C. Green M.R. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3145-3150Crossref PubMed Scopus (54) Google Scholar, A. Meisterernst M. 1996; PubMed Scopus Google Scholar), a the general transcription factors was required for of Mediator transcription about at all of not transcription to the at of NC2 in not at NC2 as have been Mediator the effect of Mediator transcription, opposing the effect of inhibitors in yeast cell effects be by Mediator. srb4ts yeast was to and the were for of basal transcription in srb4ts at The was and was by wild type Mediator but not by srb4ts Mediator not The was further on and at of and were and in the of the and of Mediator was the of the not and Not complex as by of Mediator transcription, but the of was the at all Mediator in not at Mediator as with Mediator to stimulate basal transcription to We (3Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (886) Google Scholar) and (14Mittler G. Kremmer E. Timmers H.T. Meisterernst M. EMBO Rep. 2001; 2: 808-813Crossref PubMed Scopus (103) Google Scholar, 15Baek H.J. Malik S. Qin J. Roeder R.G. Mol. Cell Biol. 2002; 22: 2842-2852Crossref PubMed Scopus (109) Google Scholar) have previously demonstrated both the stimulation of basal transcription and support of transcription by Mediator in vitro. Genetic have shown the importance of Mediator for transcription in vivo. The of the stimulation of basal transcription has an study of srb4ts Mediator this We have that both mutant cell and mutant Mediator are defective in basal transcription at an The phenotype of the srb4ts mutant may therefore be to a in basal transcription. Mediator a role in pol II transcription in vivo. We a the of Mediator in and in vivo. Mediator transcription in is required in (31Holstege F.C. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1596) Google Scholar). We that the role of Mediator in transcription is the in both but the for Mediator is in of the of transcription proteins used or of the of or the from the findings that Mediator be as a general transcription the of Mediator is all pol II are in the srb4ts strain at a restrictive the of Mediator is It transcription in a It not by suppressors of the srb4ts mutation to be an as yet of transcription effect is by Mediator. the of Mediator is required at the time as that of the well general transcription to the initiation of transcription also M. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). that Mediator of the complex with general transcription pol and Mediator to with general transcription factors M. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), at the with and initiation and the of pol of transcription J.A. S. Nature. 2000; PubMed Scopus Google Scholar). Mediator also interacts directly with pol as and have shown that Mediator the of pol the of DNA and as well F.J. Biol. PubMed Scopus Google Scholar). This has led to the idea of a of the with Mediator on the a general transcription DNA which in pol II F.J. Biol. PubMed Scopus Google Scholar). Mediator stimulate transcription its with other of the or is its effect the of by Not or other as by genetic results (32Gadbois E.L. Chao D.M. Reese J.C. Green M.R. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3145-3150Crossref PubMed Scopus (54) Google Scholar, 33Lee T.I. Wyrick J.J. Koh S.S. Jennings E.G. Gadbois E.L. Young R.A. Mol. Cell Biol. 1998; 18: 4455-4462Crossref PubMed Scopus (85) Google We have this by all at the of as yet transcriptional and have evidence for the of We two type of the stimulation of transcription, at in the of and of of in the of of Mediator be to transcription to its and this have The of Mediator transcription on its and to an in the of an activator The possibility is that Mediator the the of the or the of the Mediator might also the of the the of transcription is information to but srb4ts Mediator and of the reported here may to For the of Mediator by temperature as as the of not with transcription. This be with a role of Mediator in complex but not in or a effect of Mediator for initiation that the of Mediator. another reported an of all general transcription factors and pol II was to srb4ts mutant cell This to transcription at the restrictive temperature, that complex was not on these the of complex and transcription the Mediator mechanism. We H. for P. Tempst and E. for analysis of Not and G. for We for G. for and and D. S. C. C. D. and M. for with