TAF25p, a Non-histone-like Subunit of TFIID and SAGA Complexes, Is Essential for Total mRNA Gene Transcription in Vivo

We demonstrate, utilizing a temperature conditional mutant allele of the gene encoding TAF25p, that this non-histone-like TBP-associated factor, which is shared between the TFIID and SAGA complexes, is required for bulk mRNA gene transcription by RNA polymerase II in vivo. Immunoblotting experiments indicate that at the restrictive temperature, inactivation of TAF25p function results in a reduction of the levels of numerous TFIID and SAGA subunits, indicating its loss of function, like the histone-like TAFs, causes degradation of the constituents of these two multisubunit complexes. These data suggest that TAF25p plays a key structural role in maintaining TFIID and SAGA complex integrity. This is the first demonstration that a non-histone-like TAF is required for continuous, high level RNA polymerase II-mediated mRNA gene transcription in living yeast cells. We demonstrate, utilizing a temperature conditional mutant allele of the gene encoding TAF25p, that this non-histone-like TBP-associated factor, which is shared between the TFIID and SAGA complexes, is required for bulk mRNA gene transcription by RNA polymerase II in vivo. Immunoblotting experiments indicate that at the restrictive temperature, inactivation of TAF25p function results in a reduction of the levels of numerous TFIID and SAGA subunits, indicating its loss of function, like the histone-like TAFs, causes degradation of the constituents of these two multisubunit complexes. These data suggest that TAF25p plays a key structural role in maintaining TFIID and SAGA complex integrity. This is the first demonstration that a non-histone-like TAF is required for continuous, high level RNA polymerase II-mediated mRNA gene transcription in living yeast cells. TBP-associated factors RNA polymerase II-specific TAF temperature-sensitive wild type gene general transcription factor preinitiation complex nuclear DNA-dependent RNA polymerase II 5-fluoroorotic acid yeast extract peptone dextrose medium supplemented with adenine synthetic complete medium whole cell extract open reading frame Spt, Ada, Gcn5 acetylase complex polymerase chain reaction polyacrylamide gel electrophoresis There is ongoing debate about the exact role that TAFIIs,1 a family of conserved integral protein subunits of TFIID, play in transcription by RNA polymerase II (see Refs. 1Holstege F.C.P. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2-4Crossref PubMed Scopus (32) Google Scholar and 2Hahn S. Cell. 1998; 95: 579-582Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar for recent reviews). In contrast to initial studies, recently published reports from a number of laboratories indicate that at least a subset of TAFIIs, the so-called histone-like TAFIIs (3Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (621) Google Scholar), are absolutely essential for ongoing high level total mRNA gene transcriptionin vivo (4Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 5Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The histone-like TAFIIs examined in these experiments are of particular interest, since it was also demonstrated recently that these TAFIIs, TAF61p (histone H2B-like), TAF60p (histone H4-like), and TAF17p (histone H3-like), are present in both the TFIID and SAGA complexes (8Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates III, J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar).The data from these studies has been used to formulate a model that postulates that the histone-like TAFIIs form the structural “core” for both TFIID and SAGA complexes and when the integrity of these particular TAFIIs is compromised, then the complexes dissociate and cease to function while the resulting free subunits are rapidly degraded. At present it is not yet clear whether the RNAP II transcription requirement for histone-like TAFII function derives from the association of these TAFIIs with the TFIID, the SAGA, or both complexes. Circumstantial evidence implicates the TFIID complex though, since all of the non-TAFII-encoding genes that comprise the SAGA complex (with the exception of TRA1 (9Saleh A. Schieltz D. Ting N. McMahon S.B. Litchfield D.W. Yates III, J.R. Lees-Miller S.P. Cole M.D. Brandl C.J. J. Biol. Chem. 1998; 273: 26559-26565Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar)) are non-essential genes, while (with but one exception, TAF30) all of the TFIID TAFIIs are encoded by essential genes. This question remains to be formally tested though.Having previously cloned and characterized TAF25, we decided to attempt to generate temperature-conditional alleles of theTAF25 gene, which we could use as a tool to examine the role of TAF25p in RNAP II transcription. We felt it important to test whether or not the non-histone-like TAFII, TAF25p, which is present in both TFIID and SAGA, is globally involved in total mRNA gene transcription by RNA polymerase II in vivo. We successfully generated temperature-conditional alleles ofTAF25 and one particular mutant allele caused yeast cells to rapidly cease growth at the restrictive temperature. We characterized this taf25 ts mutant in detail with regards to its effects both upon specific and global mRNA gene transcription and TFIID/SAGA subunit protein integrity. Contrary to the TFIID functional and structural organization models recently described by others (reviewed in Ref. 2Hahn S. Cell. 1998; 95: 579-582Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we found that TAF25p function is continuously required for high level mRNA gene transcriptionin vivo. This is the first such demonstration that a non-histone-like TAFII protein is continuously required for mRNA gene transcription in vivo. We discuss how these new data regarding TAF25p may be incorporated into current models of TFIID structural organization and function. There is ongoing debate about the exact role that TAFIIs,1 a family of conserved integral protein subunits of TFIID, play in transcription by RNA polymerase II (see Refs. 1Holstege F.C.P. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2-4Crossref PubMed Scopus (32) Google Scholar and 2Hahn S. Cell. 1998; 95: 579-582Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar for recent reviews). In contrast to initial studies, recently published reports from a number of laboratories indicate that at least a subset of TAFIIs, the so-called histone-like TAFIIs (3Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (621) Google Scholar), are absolutely essential for ongoing high level total mRNA gene transcriptionin vivo (4Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 5Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The histone-like TAFIIs examined in these experiments are of particular interest, since it was also demonstrated recently that these TAFIIs, TAF61p (histone H2B-like), TAF60p (histone H4-like), and TAF17p (histone H3-like), are present in both the TFIID and SAGA complexes (8Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates III, J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). The data from these studies has been used to formulate a model that postulates that the histone-like TAFIIs form the structural “core” for both TFIID and SAGA complexes and when the integrity of these particular TAFIIs is compromised, then the complexes dissociate and cease to function while the resulting free subunits are rapidly degraded. At present it is not yet clear whether the RNAP II transcription requirement for histone-like TAFII function derives from the association of these TAFIIs with the TFIID, the SAGA, or both complexes. Circumstantial evidence implicates the TFIID complex though, since all of the non-TAFII-encoding genes that comprise the SAGA complex (with the exception of TRA1 (9Saleh A. Schieltz D. Ting N. McMahon S.B. Litchfield D.W. Yates III, J.R. Lees-Miller S.P. Cole M.D. Brandl C.J. J. Biol. Chem. 1998; 273: 26559-26565Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar)) are non-essential genes, while (with but one exception, TAF30) all of the TFIID TAFIIs are encoded by essential genes. This question remains to be formally tested though. Having previously cloned and characterized TAF25, we decided to attempt to generate temperature-conditional alleles of theTAF25 gene, which we could use as a tool to examine the role of TAF25p in RNAP II transcription. We felt it important to test whether or not the non-histone-like TAFII, TAF25p, which is present in both TFIID and SAGA, is globally involved in total mRNA gene transcription by RNA polymerase II in vivo. We successfully generated temperature-conditional alleles ofTAF25 and one particular mutant allele caused yeast cells to rapidly cease growth at the restrictive temperature. We characterized this taf25 ts mutant in detail with regards to its effects both upon specific and global mRNA gene transcription and TFIID/SAGA subunit protein integrity. Contrary to the TFIID functional and structural organization models recently described by others (reviewed in Ref. 2Hahn S. Cell. 1998; 95: 579-582Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we found that TAF25p function is continuously required for high level mRNA gene transcriptionin vivo. This is the first such demonstration that a non-histone-like TAFII protein is continuously required for mRNA gene transcription in vivo. We discuss how these new data regarding TAF25p may be incorporated into current models of TFIID structural organization and function. We thank our laboratory colleagues for their constructive criticism and advice throughout the course of these studies as well as for their generous sharing of reagents and strains. We thank K. Struhl, J. Haber, and R. Young for gifts of strains and plasmids. We also thank S. Berger and D. Allis for providing Ada2p and Gcn5p E. coli expression plasmids.