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Requirement for Yeast TAF145 Function in Transcriptional Activation of the RPS5 Promoter That Depends on Both Core Promoter Structure and Upstream Activating Sequences

2001; Elsevier BV; Volume: 276; Issue: 28 Linguagem: Inglês

10.1074/jbc.m102416200

ISSN

1083-351X

Autores

Yoshihiro Tsukihashi, Masashi Kawaichi, Tetsuro Kokubo,

Tópico(s)

RNA Research and Splicing

Resumo

The general transcription factor TFIID has been shown to be involved in both core promoter recognition and the transcriptional activation of eukaryotic genes. We recently isolated TAF145 (one of TFIID subunits) temperature-sensitive mutants in yeast, in which transcription of the TUB2 gene is impaired at restrictive temperatures due to a defect in core promoter recognition. Here, we show in these mutants that the transcription of theRPS5 gene is impaired, mostly due to a defect in transcriptional activation rather than to a defect in core promoter recognition, although the latter is slightly affected as well. Surprisingly, the RPS5 core promoter can be activated by various activation domains fused to a GAL4 DNA binding domain, but not by the original upstream activating sequence (UAS) of theRPS5 gene. In addition, a heterologous CYC1core promoter can be activated by RPS5-UAS at normal levels even in these mutants. These observations indicate that a distinct combination of core promoters and activators may exploit alternative activation pathways that vary in their requirement for TAF145 function. In addition, a particular function of TAF145 that is deleted in our mutants appears to be involved in both core promoter recognition and transcriptional activation. The general transcription factor TFIID has been shown to be involved in both core promoter recognition and the transcriptional activation of eukaryotic genes. We recently isolated TAF145 (one of TFIID subunits) temperature-sensitive mutants in yeast, in which transcription of the TUB2 gene is impaired at restrictive temperatures due to a defect in core promoter recognition. Here, we show in these mutants that the transcription of theRPS5 gene is impaired, mostly due to a defect in transcriptional activation rather than to a defect in core promoter recognition, although the latter is slightly affected as well. Surprisingly, the RPS5 core promoter can be activated by various activation domains fused to a GAL4 DNA binding domain, but not by the original upstream activating sequence (UAS) of theRPS5 gene. In addition, a heterologous CYC1core promoter can be activated by RPS5-UAS at normal levels even in these mutants. These observations indicate that a distinct combination of core promoters and activators may exploit alternative activation pathways that vary in their requirement for TAF145 function. In addition, a particular function of TAF145 that is deleted in our mutants appears to be involved in both core promoter recognition and transcriptional activation. TATA-binding protein TBP-associated factor upstream activating sequence DNA binding domain polymerase chain reaction base pair(s) In eukaryotes, transcriptional initiation by RNA polymerase II requires a set of general transcriptional factors (reviewed in Refs. 1Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 2Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Crossref PubMed Google Scholar, 3Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (851) Google Scholar). These factors are assembled in a stepwise manner to form a preinitiation complex on the core promoter (1Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar) or are recruited as a few preassembled units (4Koleske A.J. Young R.A. Trends Biochem. Sci. 1995; 20: 113-116Abstract Full Text PDF PubMed Scopus (267) Google Scholar, 5Ptashne M. Gann A. Nature. 1997; 386: 569-577Crossref PubMed Scopus (945) Google Scholar, 6Ranish J.A. Yudkovsky N. Hahn S. Genes Dev. 1999; 13: 49-63Crossref PubMed Scopus (206) Google Scholar). In either case, the first step in preinitiation complex assembly is the binding of a protein complex called TFIID to the core promoter, which in turn provides a structural platform for the remainder of the general transcriptional factors to be incorporated (6Ranish J.A. Yudkovsky N. Hahn S. Genes Dev. 1999; 13: 49-63Crossref PubMed Scopus (206) Google Scholar). Previous studies have shown that TFIID-promoter interactions are a major rate-limiting step during transcriptional initiation and therefore are one of the most important molecular targets for transcriptional activators (7Lieberman P.M. Berk A.J. Genes Dev. 1994; 8: 995-1006Crossref PubMed Scopus (192) Google Scholar, 8Chi T. Carey M. Genes Dev. 1996; 10: 2540-2550Crossref PubMed Scopus (118) Google Scholar, 9Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholar). TFIID is a multiprotein complex composed of the TATA-binding protein (TBP)1 and ∼10–12 phylogenetically conserved TBP-associated factors (TAFs) (reviewed in Refs. 9Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholar and 10Albright S.R. Tjian R. Gene. 2000; 242: 1-13Crossref PubMed Scopus (275) Google Scholar). A number of biochemical studies have revealed coactivator and core promoter recognition activities to be two important functions for TAFs (reviewed in Refs. 9Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholar, 10Albright S.R. Tjian R. Gene. 2000; 242: 1-13Crossref PubMed Scopus (275) Google Scholar, 11Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Earlier experiments using in vitro transcription systems demonstrated that TBP can mediate basal transcription but is unable to support activated transcription by itself. In contrast, TFIID, even when reconstituted with recombinant TBP and TAFs (12Chen J.L. Attardi L.D. Verrijzer C.P. Yokomori K. Tjian R. Cell. 1994; 79: 93-105Abstract Full Text PDF PubMed Scopus (328) Google Scholar), mediates both basal and activated transcription, supporting the idea that TAFs are essential cofactors for transcriptional activation (reviewed in Refs. 9Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholarand 10Albright S.R. Tjian R. Gene. 2000; 242: 1-13Crossref PubMed Scopus (275) Google Scholar). More recent studies have begun to address how core promoters of eukaryotic genes are recognized by TFIID (reviewed in Refs. 13Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (180) Google Scholar and14Kutach A.K. Kadonaga J.T. Mol. Cell. Biol. 2000; 20: 4754-4764Crossref PubMed Scopus (274) Google Scholar). The three classes of core promoter elements that are currently known are the TATA element, the initiator, and the downstream promoter element, each of which may serve as a recognition site for distinct TFIID subunits (reviewed in Refs. 13Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (180) Google Scholar and 14Kutach A.K. Kadonaga J.T. Mol. Cell. Biol. 2000; 20: 4754-4764Crossref PubMed Scopus (274) Google Scholar). In addition to the extensively characterized TBP-TATA element interactions (15Patikoglou G.A. Kim J.L. Sun L. Yang S.H. Kodadek T. Burley S.K. Genes Dev. 1999; 13: 3217-3230Crossref PubMed Scopus (246) Google Scholar), the initiator and downstream promoter element have been shown to be recognized by TAF250-TAF150 and TAF60-TAF40 heterodimers, respectively (13Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (180) Google Scholar, 16Burke T.W. Kadonaga J.T. Genes Dev. 1997; 11: 3020-3031Crossref PubMed Scopus (400) Google Scholar). These TAF-DNA and TBP-DNA interactions are important for the ability of TFIID to bind to the core promoter specifically and to mediate transcription efficiently (reviewed in Refs. 13Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (180) Google Scholar and 14Kutach A.K. Kadonaga J.T. Mol. Cell. Biol. 2000; 20: 4754-4764Crossref PubMed Scopus (274) Google Scholar). In addition, other cofactors such as TFIIA (17Martinez E. Ge H. Tao Y. Yuan C.X. Palhan V. Roeder R.G. Mol. Cell. Biol. 1998; 18: 6571-6583Crossref PubMed Scopus (62) Google Scholar), TAFII- and initiator-dependent cofactors (17Martinez E. Ge H. Tao Y. Yuan C.X. Palhan V. Roeder R.G. Mol. Cell. Biol. 1998; 18: 6571-6583Crossref PubMed Scopus (62) Google Scholar), and NC2 (18Willy P.J. Kobayashi R. Kadonaga J.T. Science. 2000; 290: 982-985Crossref PubMed Scopus (135) Google Scholar), appear to modulate the recognition by TFIID of a wide range of core promoter structures. These principal functions of TAFs have also been evaluated in living cells (reviewed in Refs. 10Albright S.R. Tjian R. Gene. 2000; 242: 1-13Crossref PubMed Scopus (275) Google Scholar and 11Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Genetic depletion or inactivation analysis of yeast TAF145, a subunit that is orthologous to human TAF250, revealed that it was not required for transcription generally but was essential for a subset of genes in vivo (reviewed in Ref. 11Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Promoter swapping experiments demonstrated that TAF145 function is demanded by the core promoter region rather than by upstream activating sequences (UASs) as examined for theCLN2, RPS5, and TUB2 genes (19Shen W.-C. Green M.R. Cell. 1997; 90: 615-624Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). Thus, it appears that TAF145 function is tightly connected to core promoter recognition, in accordance with human TAF250, which directly recognizes an initiator element as described above (13Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (180) Google Scholar). On the other hand, TAF145 has been shown to be required for other transcriptional activities, such as activation of the ADH2 gene by Adr1 (21Komarnitsky P.B. Klebanow E.R. Weil P.A. Denis C.L. Mol. Cell. Biol. 1998; 18: 5861-5867Crossref PubMed Scopus (21) Google Scholar) and derepression of RNR genes by DNA damage, that are normally repressed by the Crt1 and Tup1-Ssn6 corepressor complex (22Li B. Reese J.C. EMBO J. 2000; 19: 4091-4100Crossref PubMed Scopus (28) Google Scholar). Consistent with such apparently broad roles in transcription, yeast TAF145 and/or human TAF250 possess multiple activities (e.g.TAF N-terminal domain activity, which inhibits TBP function (23Kotani T. Banno K. Ikura M. Hinnebusch A.G. Nakatani Y. Kawaichi M. Kokubo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7178-7183Crossref PubMed Scopus (40) Google Scholar,24Kotani T. Miyake T. Tsukihashi Y. Hinnebusch A.G. Nakatani Y. Kawaichi M. Kokubo T. J. Biol. Chem. 1998; 273: 32254-32264Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar); serine/threonine kinase that autophosphorylates and transphosphorylates TFIIF (25O'Brien T. Tjian R. Mol Cell. 1998; 1: 905-911Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar); histone acetyl transferase, which acetylates histones and TFIIE (26Mizzen C.A. Yang X.J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 27Imhof A. Yang X.J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar); two bromodomains, which bind acetylated histones (28Jacobson R.H. Ladurner A.G. King D.S. Tjian R. Science. 2000; 288: 1422-1425Crossref PubMed Scopus (680) Google Scholar); and a ubiquitin-activating/conjugating activity for histone H1 (29Pham A.D. Sauer F. Science. 2000; 289: 2357-2360Crossref PubMed Scopus (203) Google Scholar)). Some of these activities have been shown to be required for gene expression in vivo (29Pham A.D. Sauer F. Science. 2000; 289: 2357-2360Crossref PubMed Scopus (203) Google Scholar, 30O'Brien T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2456-2461Crossref PubMed Scopus (53) Google Scholar, 31Dunphy E.L. Johnson T. Auerbach S.S. Wang E.H. Mol. Cell. Biol. 2000; 20: 1134-1139Crossref PubMed Scopus (61) Google Scholar). How broadly TAF functions are required for gene expression has been extensively studied in yeast (reviewed in Refs. 11Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 32Holstege 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 (1598) Google Scholar, and 33Lee T.I. Causton H.C. Holstege F.C. Shen W.C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (302) Google Scholar). Genome-wide expression analysis suggests that any TAFs thus far examined are not universally required for transcription, in contrast to other general transcriptional factors, such as Srb4, Kin28, and the largest subunit of RNA polymerase II, which are required for almost all genes (reviewed in Refs. 11Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 32Holstege 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 (1598) Google Scholar, and 33Lee T.I. Causton H.C. Holstege F.C. Shen W.C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (302) Google Scholar). More puzzlingly, mutations in different TAFs affect different sets of genes, ranging from 3% (tsm1) to 67% (taf17) of the whole genome (33Lee T.I. Causton H.C. Holstege F.C. Shen W.C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (302) Google Scholar). It has been proposed that TAFs shared by TFIID and SAGA, another crucial histone acetyl transferase complex regulating a subset of genes distinct from TFIID (34Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates J.r. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar), tend to affect a broader range of genes. This is probably because mutations in the TAFs that they have in common should decrease the activities of TFIID and SAGA simultaneously (reviewed in Ref. 35Hahn S. Cell. 1998; 95: 579-582Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, this supposition has not yet been firmly established, because TFIID-specific TAFs such as TAF40 (36Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Crossref PubMed Scopus (53) Google Scholar) and TAF48/TSG2 (37Reese J.C. Zhang Z. Kurpad H. J. Biol. Chem. 2000; 275: 17391-17398Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 38Sanders S.L. Weil P.A. J. Biol. Chem. 2000; 275: 13895-13900Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) have been shown to be generally required for transcription. The wide range of affected genes (3–67% of the genome) may be partly explained by the allele specificities because, for example, the tighter allele of TAF17 caused much more dramatic loss of transcription than the milder one (39Michel B. Komarnitsky P. Buratowski S. Mol Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The recent finding that chick TAF31 can be deleted genetically without affecting the expression of most genes, although it is an ortholog of the TAF (TAF17) that has the most universal function among yeast TAFs, indicates that the requirement for TAFs is evolutionarily divergent (40Chen Z. Manley J.L. Mol. Cell. Biol. 2000; 20: 5064-5076Crossref PubMed Scopus (36) Google Scholar). To further clarify how TAF function is involved in gene expressionin vivo, we believe it is important to isolate a wide range of conditional taf alleles and to inspect the transcriptional defect at the molecular level in each tafmutant. We recently isolated two novel temperature-sensitivetaf145 mutants in which the expression profiles of some genes were not identical to those in previously reportedtaf145 mutants (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). In our mutants, the core promoter of theTUB2 gene failed to mediate basal transcription, but the function was restored by inserting a consensus TATA element (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). Consistent with this, we show here that the TATA element is important for transcription from the CLN2 and CYC1promoters. Interestingly, however, the creation of a consensus TATA element cannot restore transcription from the RPS5 promoter. We demonstrate that the RPS5 promoter is mostly impaired in activated transcription and only slightly impaired in basal transcription and that the creation of a consensus TATA element was able to rescue the latter defect. Most importantly, we find that the requirement for TAF145 function in activated transcription of theRPS5 promoter depends on both core promoter structure and UASs. These results imply that a specific function of TAF145 is involved in both core promoter recognition and core promoter and UAS-specific activated transcription. Standard techniques were used for yeast growth and transformation (41Lundblack V. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1998: 13.0.1-13.13.9Google Scholar, 42Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar). The yeast strains used in this study, YTK3010 (wild type), YTK3002 (Y570N), YTK3003 (N568Δ), and YTK3005 (T657K) were generated by plasmid shuffle techniques from the parental strain Y22.1 (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). They carry a deletion of the chromosomal TAF145 coding region and the wild type or mutantTAF145 gene on a TRP1-based low copy number vector (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). pM1452 (shown as ΔCLN2 TATA in Fig. 1) and pM1591 (shown asUAS GAL +CYC1 TATA /–174in Fig. 1 and UAS GAL +CYC1/–174 in Fig.5) were described previously (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). pM1452 and pM1591 were subjected to site-specific mutagenesis (43Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4560) Google Scholar) to create pM3226 (shown asΔCLN2 GAGA in Fig. 1) and pM3227 (shown as UAS GAL+CYC1 GAGA/–174 in Fig.1) using oligonucleotides TK1289 and TK1290, respectively. The oligonucleotides used in this study are listed in TableI. pM3228 (shown as RPS5/–593 TAAAAT in Fig. 1) was constructed by replacing the 765-bpSphI/XhoI fragment of pM1452 (encompassing theCLN2 promoter) with a 732-bp DNA fragment containing theRPS5 promoter, which was amplified by PCR using the primer pair TK1291 (+SphI)-TK1292 (+XhoI) and genomic DNA as a template. pM3228 was subsequently subjected to site-specific mutagenesis to create pM3229 (TATAAA), pM3230 (TATAAAAA), pM3231 (TATATAAA), and pM3232 (TATATAAAAA), using the oligonucleotides TK1293, TK1466, TK1467, and TK1468, respectively.Figure 5TAF145 dependence was observed only when theRPS5 UAS was combined with the RPS5core promoter in taf145 mutants. A, schematic representation of the reporter plasmids used in this experiment. The regions derived from the CLN2,RPS5, and CYC1 genes are shown by open, shaded, and striped boxes, respectively.RPS5-UAS was fused with the RPS5 andCYC1 core promoters in its two forms, i.e.UAS90bp (–450∼–361 bp) or UAS150bp(–450∼–300 bp), to generate UAS90bp+RPS5/–87, UAS150bp+RPS5/–200, UAS150bp+RPS5/–87, and UAS150bp+CYC1/–174. Synthetic binding sites for GAL4 fusion activators were linked upstream of the CYC1 andRPS5 core promoters to generate UASGAL+CYC1/–174 and UASGAL+RPS5/ –87C, respectively. B andC, Northern blot analysis of mRNA with aCLN2-specific probe. Wild type or mutant strains carrying the indicated reporter plasmids were cultured at 25 or 37 °C. Total RNA isolated from these cultures was analyzed as described in Fig. 1. In C, activator expression plasmids (GAL4DBD-RAP1, -VP16C, -EBNA2, and -GCN4, or GAL4DBD alone as a negative control) were introduced into yeast cells with the reporter plasmid as indicated to measure the activation efficiencies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IOligonucleotides used in this studyNo.SequenceTK11365′-CAC ACT AGT CAG ATC CGC CAG GCG TGT-3′TK11375′-CAC CTC GAG CCT TAT GTG GGC CAC CCT-3′TK12895′-GAT TGC TAT TTT TTT CTC TAT GAA AGA GAT AGT-3′TK12905′-GAA AAC AAG AGT TTT CTC TAC ATA CAG AGC ACA-3′TK12915′-CAC GCA TGC GAT TTC ACG AAA ACA CCT-3′TK12925′-CAC CTC GAG GGA CTT CTT CTG GAA TTG-3′TK12935′-TAA AAA GTA CAA AAT TTT ATA GAA CTA ATG GGC A-3′TK13215′-CAC ACT AGT ACT TTT TAT CAA TAC TTA-3′TK13225′-AAT AGT TAC CAG AGT ACT AGT CTA TTT TTT TAT ATA-3′TK14355′-CAC GCA TGC ACT ATC TCT TTC ATA TAT-3′TK14365′-CAC GCA TGC GCA CGC AGC CTC TGG CTA-3′TK14665′-GA TAA AAA GTA CAA ATT TTT ATA GAA CTA ATG GGC A-3′TK14675′-TAA AAA GTA CAA AAT TTT ATA TA GAA CTA ATG GGC ACA-3′TK14685′-GA TAA AAA GTA CAA ATT TTT ATA TA GAA CTA ATG GGC ACA-3′TK15005′-CAC GCA TGC TCT CTT TCA TAT ATA AAA-3′TK15015′-CAC GCA TGC TTT CAT ATA TAA AAA AAT-3′TK15025′-CAC GCA TGC TCA TAT ATA AAA AAA TAG-3′TK15035′-CAC GCA TGC ATA TAT AAA AAA ATA GAC-3′TK15045′-CAC GCA TGC ATA TAA AAA AAT AGA CTA-3′TK15055′-CAC GCA TGC ATA AAA AAA TAG ACT AGT-3′TK15415′-CCT AAA ATA TTT TGT A-3′TK15425′-CTA GTA CAA AAT ATT TTA GGC ATG-3′TK15435′-CAT AAA ATA TTT TGT A-3′TK15445′-CTA GTA CAA AAT ATT TTA TGC ATG-3′TK15455′-CCT ATA AAA TTT TGT A-3′TK15465′-CTA GTA CAA AAT TTT ATA GGC ATG-3′TK15475′-CAT ATA AAA TTT TGT A-3′TK15485′-CTA GTA CAA AAT TTT ATA TGC ATG-3′TK15495′-CAC GCA TGC CTA AAA TAT TTT GTA CTT-3′TK15505′-CAC GCA TGC CTA TAA AAT TTT GTA CTT-3′TK15515′-CAC GCA TGC ATA AAA TAT TTT GTA CTT-3′TK15525′-CAC GCA TGC ATA TAA AAT TTT GTA CTT-3′TK16245′-CAC GCA TGC TTA TAC AAC AAC ACC CAT-3′TK16255′-CAC GCA TGC AGA ATT CTT TTT CTC CCG-3′TK16265′-CAC GCA TGC CTC CCC TAC CTT CGC CGC-3′TK16275′-CAC GCA TGC GCA GCC CGG GGG ATC CAC TAG T-3′TK16285′-CAC GCA TGC ACC GCG GTG GCG GCC GCT-3′TK16945′-CAC GAA TTC TCT TAC GCT ATA CCA GAA-3′TK16955′-CAC CTG CAG CTA GCT TAT GGT ATC AGG ATC-3′TK16965′-TAA ACT TGA GAC AAA AGA ATA ATT ATG GGT-3′TK16975′-CAC GCA TGC GAT GCA GGG CCA TTC TCA-3′TK17335′-CAC GCA TGC CCT TCC CCG TAG CAG GGC-3′TK17345′-CAC ACT AGT TTA TAC AAC AAC ACC CAT-3′TK17355′-CAC ACT AGT CCT TCC CCG TAG CAG GGC-3′ Open table in a new tab To enable the construction of CLN2-RPS5 hybrid promoters, aSpeI site was created at the –68 bp position of pM1452 by site-specific mutagenesis, using the TK1322 oligonucleotide. The 506-bpSpeI/XhoI fragment containing the CLN2initiator of the resulting plasmid pM3233 was subsequently replaced with the 212-bp DNA fragment containing the RPS5 initiator, which was amplified by PCR using the primer pair TK1321 (+SpeI)-TK1292 (+XhoI) and pM3228 (RPS5/–593) as a template, to create pM3234 (shown as CLN2(–332)-RPS5 in Fig. 2). For deletion analysis of the CLN2-RPS5 hybrid promoter, pM3235 (CLN2(–126)-RPS5 in Fig.2), pM3236 (CLN2(–96)-RPS5in Fig. 2), pM3237 (CLN2(–92)-RPS5 in Fig. 2), pM3238 (CLN2(–88)-RPS5 in Fig. 2), pM3239 (CLN2(–86)-RPS5 in Fig. 2), pM3240 (CLN2(–84)-RPS5 in Fig. 2), pM3241 (CLN2(–82)-RPS5 in Fig. 2), and pM3242 (CLN2(–80)-RPS5in Fig. 2) were constructed by replacing the 476-bpSphI/XhoI fragment of pM3234 with DNA fragments encoding the –126 (CLN2)∼+139 (RPS5), –96 (CLN2)∼+139 (RPS5), –92 (CLN2)∼+139 (RPS5), –88 (CLN2)∼+139 (RPS5), –86 (CLN2)∼+139 (RPS5), –84 (CLN2)∼+139 (RPS5), –82 (CLN2)∼+139 (RPS5), and –80 (CLN2)∼+139 (RPS5) fragments of theCLN2-RPS5 hybrid promoter, respectively; these fragments were amplified by PCR using pM3234 (CLN2(–332)-RPS5) as a template and the following primer pairs: TK1436-TK1292, TK1435-TK1292, TK1500-TK1292, TK1501-TK1292, TK1502-TK1292, TK1503-TK1292, TK1504-TK1292, and TK1505- TK1292. pM3244 (shown as RPS5/–87C in Fig. 3), pM3245 (RPS5 TATAAA /–87C in Fig. 3), pM3248 (RPS5/–87A in Fig. 3), and pM3249 (RPS5 TATAAA /–87A in Fig. 3) were constructed by replacing the 765-bp SphI/XhoI fragment of pM1452 with the DNA fragments containing theRPS5 core promoter or its derivatives that were amplified by PCR using the primer pairs TK1549-TK1292, TK1550-TK1292, TK1551-TK1292, and TK1552-TK1292 and pM3228 (RPS5/–593) as a template. pM3246 (RPS5/–87C+SpeI in Fig. 3), pM3247 (RPS5TATAAA/–87C+SpeI in Fig. 3), pM3250 (RPS5/–87A+SpeI), and pM3251 (RPS5TATAAA/–87A+SpeI in Fig. 3) were constructed by replacing the 264-bp SphI/SpeI fragment of pM3234 (CLN2(–332)-RPS5in Fig. 2) with the short DNA fragments generated by annealing two oligonucleotide pairs, TK1541-TK1542, TK1545-TK1546, TK1543-TK1544, and TK1547-TK1548, respectively. For deletion analysis of the RPS5 promoter (Fig. 4), pM3252 (RPS5/–450 in Fig. 4), pM3253 (RPS5/–300 in Fig. 4), and pM3254 (RPS5/–200 in Fig. 4) were constructed by replacing the 765-bp SphI/XhoI fragment of pM1452 with DNA fragments encoding the –450∼+139-, –300∼+139-, and –200∼+139-bp regions of the RPS5 promoter, respectively; these fragments were amplified by PCR using the primer pairs TK1624-TK1292, TK1625-TK1292, and TK1626-TK1292, respectively, and pM3228 (RPS5/–593) as a template. pM3255 (RPS5/–593/ΔUASRAP1 in Fig. 4) was generated by removing the presumptive RAP1 binding site (–403∼–415 bp) (44Lascaris R.F. Mager W.H. Planta R.J. Bioinformatics. 1999; 15: 267-277Crossref PubMed Scopus (80) Google Scholar) of pM3228 (RPS5/–593) by site-specific mutagenesis using the oligonucleotide TK1696. pM3256 (shown as UAS 90bp +RPS5/–87 in Fig. 5) was created by ligating the 90-bp DNA fragment encoding –450∼–361-bp of RPS5-UAS, which was amplified by PCR using the primer pair TK1624-TK1697 and pM3228 (RPS5/–593) as a template, into the SphI site of pM3244 (RPS5/–87C). Similarly, pM3257 (UAS 150bp +RPS5/–200 in Fig. 5) and pM3258 (UAS 150bp +RPS5/–87 in Fig. 5) were created by ligating the 150-bp (–450∼–300 bp of RPS5-UAS) DNA fragment amplified by the primer pair TK1624-TK1733 into theSphI sites of pM3254 (RPS5/–200) and pM3244 (RPS5/–87C), respectively. To construct pM3259 (UAS150bp+CYC1/–174 in Fig. 5), pM1588 was created first by replacing the 260-bpSpeI/XhoI fragment of pM1585 (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar) with the 344-bp PCR fragment containing the CYC1 promoter amplified by the primer pair TK1136-TK1137. pM3259 was subsequently created by ligating the 150-bp (–450∼–300 bp of RPS5-UAS) DNA fragment amplified by the primer pair TK1734-TK1735 into the SpeI site of pM1588. pM3260 was created by ligating the DNA fragment containing four repeats of the GAL4 binding site, which was amplified by PCR using the primer pair TK1627-TK1628 and pM2190 as a template, into the SphI site of pM3244 (RPS5/–87C). The PvuII fragment of pM3266 containing the entire reporter gene was moved into pRS316 (45Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to change the auxotrophic marker from LEU2 to URA3. The resulting plasmid pM3279 is shown asUAS GAL +RPS5/–87C in Fig. 5. The plasmids expressing activators in yeast cells, pM524 (GAL4DBD-VP16C (amino acids 457–490)), pM1570 (GAL4DBD-EBNA2 (amino acids 426–462)), pM967 (GAL4DBD-GCN4 (amino acids 107–144)), and pM471 (GAL4DBD) have been described previously (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). pM3261 (GAL4DBD-RAP1 (amino acids 630–690)) was similarly constructed by ligating the DNA fragment encoding the RAP1 activation domain (amino acids 630–690) (46Graham I.R. Haw R.A. Spink K.G. Halden K.A. Chambers A. Mol. Cell. Biol. 1999; 19: 7481-7490Crossref PubMed Scopus (41) Google Scholar) that was amplified using the primer pair TK1694-TK1695 into the EcoRI/PstI sites of pM471. Northern blot analyses were performed as described previously (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). All mRNAs derived from mini-CLN2 hybrid gene reporter constructs were detected by the 32P-labeled 411-bp XhoI/HindIII fragment isolated from pM1452. We previously demonstrated that transcription of a subset of genes at 37 °C is drastically impaired in the taf145-N568Δ and –T657K mutants but only slightly impaired in the taf145-Y570N mutant, despite the much slower growth phenotypes of all of these mutants at 37 °C (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000; 20: 2385-2399Crossref PubMed Scopus (36) Google Scholar). Interestingly, the impaired transcription from theTUB2 promoter in the former two mutants can be rescued by creating a consensus TATA element, indicating that the TATA element compensates for the loss of TAF145 function (20Tsukihashi Y. Miyake T. Kawaichi M. Kokubo T. Mol. Cell. Biol. 2000

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