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1 Division of Molecular and Cellular Biology, Graduate School of Integrated Science, Yokohama City University, Yokohama, 230-0045, Japan
2
Division of Gene Function in Animals, Nara Institute of Science and Technology, Ikoma, 630-0192, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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It has been proposed that activators recruit TFIID on to the core promoter (Albright & Tjian 2000; Roeder 1998). In fact, upstream activating sequences (UAS) in the promoters of ribosomal protein genes efficiently recruit TFIID to several core promoters (Li et al. 2002; Mencia et al. 2002). However, when a TAF-LexA fusion protein is used to recruit TFIID to a promoter with a LexA binding site, transcription is less efficient than in the presence of a LexA-Gal4 activator (Gaudreau et al. 1999). Thus, TFIID may not be sufficient to fully activate transcription. It has been suggested that to achieve full activation, TFIID must undergo a conformational change that removes an intrinsic inhibitor of activation and allows stable core promoter binding (Chi & Carey 1996; Guermah et al. 2001; Lieberman & Berk 1994; Ozer et al. 1998; Sanders et al. 2002).
TAF1 protein (Taf1p) is a well characterized TAF that inhibits binding of TBP to TATA elements. The inhibitory function is contained in the Taf1p N-terminal domain (TAND) (Bai et al. 1997; Kokubo et al. 1994, 1998; Kotani et al. 1998), which includes the TAND1 and TAND2 subdomains. TAND1 and TAND2 bind the concave and convex surfaces of TBP and there compete with activators and TFIIA, respectively (Kokubo et al. 1998; Kotani et al. 1998; Liu et al. 1998; Nishikawa et al. 1997). A two-step handoff model has been proposed for TFIID activation: step 1 involves transfer of TBP from TAND to an activator and step 2 delivers TBP to the TATA element (Kotani et al. 2000). Genetic studies provide indirect evidence to support this hypothesis (Kobayashi et al. 2001, 2003).
Recent studies also provide evidence that TAND regulates transcription in vivo. For example, experiments with human factors suggest that c-Jun (Lively et al. 2001) and NF-
B (Guermah et al. 1998) activate transcription, at least in part, by relieving the inhibitory effect of TAND. In yeast, a Taf1p mutant with a TAND1 deletion stimulates transcription more efficiently than intact Taf1p (Cheng et al. 2002). Furthermore, a deficiency of the global transcriptional regulator Ccr4-Not was rescued by deletion of TAND2, but not TAND1, suggesting that TAND1 and TAND2 are not functionally equivalent (Deluen et al. 2002). A genome-wide expression analysis has also suggested that TAND and TBP play interdependent roles in regulating transcription of some genes in vivo (Chitikila et al. 2002). These observations strongly suggest that TAND plays an important role in regulating TFIID-dependent transcription.
Earlier studies showed that yeast TAND1 (yTAND1: amino acids [aa] 10-37) and TAND2 (yTAND2: aa 46-71) could be functionally replaced with their Drosophila counterparts, dTAND1 and dTAND2, in vitro (Kotani et al. 1998). However, the chimeric Taf1p with dTAND was unstable in vivo; a low level of full-length Taf1p and smaller truncated forms of chimeric Taf1p were produced when chimeric Taf1p was expressed in yeast (Kotani et al. 1998). The goal of this study was to determine the mechanism causing chimeric Taf1p to be truncated in vivo and to investigate whether wild-type (WT) yeast Taf1p is sensitive to the same process. The results indicate that TAND is deleted from chimeric Taf1p in vivo by a complex mechanism involving alternative transcription and/or translation initiation sites. Importantly, the same mechanisms produce a small amount of TAND-deficient Taf1p in WT yeast. This study also shows that dTAND2 substitutes functionally for yTAND2.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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Earlier studies showed that yeast TAND1 (yTAND1) and TAND2 (yTAND2) could be replaced with their Drosophila counterparts, dTAND1 and dTAND2 in vitro, but the chimeric proteins are truncated when expressed in yeast cells (Kotani et al. 1998). 
taf1 yeast cells expressing chimeric Taf1p are temperature-sensitive (TS) for growth, suggesting either that dTAND domains do not substitute functionally for yTAND domains, or that truncated forms of chimeric Taf1p are toxic to the cell. This study examines the mechanism(s) underlying these observations. Note that in this manuscript, Taf1p with dTAND1 is called d1y2, Taf1p with dTAND2 is called y1d2 and WT Taf1p is called y1y2.
The structure of truncated chimeric Taf1p was characterized using variants of chimeric Taf1p tagged at the amino- or carboxy-terminus with haemagglutinin (HA). Whole cell lysates were prepared from d1y2, y1d2, WT (y1y2) and TAND strains and analysed by immunoblot (Fig. 1A). The anti-Taf1p and anti-HA immunoreactivities were similar for Taf1p tagged with HA at the carboxy-terminus but not for Taf1p tagged at the amino-terminus (Fig. 1A). This result suggests that truncated d1y2 and y1d2 are N-terminally deleted and may lack all or part of TAND.
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TAND Taf1p are also detected (Fig. 1A, lanes 13, 14) (see also below).
The amino-terminal deletions in the d1y2 and y1d2 strains were more precisely mapped using recombinant amino-terminally truncated mutants of Taf1p as size markers (Fig. 1B). The most prominent truncated proteins migrated in the same region of the gel as deletion mutants 2-86/2-96/2-106 and 2-176/2-186/2-196, suggesting that the largest truncated form of chimeric Taf1p lacks approximately residues 1-96 and the smallest truncated form of chimeric Taf1p lacks approximately residues 1-186.
DNA sequences required for truncation of Taf1p TAND chimeras
The results described above indicate that similar amino-terminally truncated forms of Taf1p accumulate in yeast cells expressing WT (y1y2) or chimeric (d1y2, y1d2) TAND (e.g. compare lanes 13-16 in Fig. 1A), but the truncated forms of the chimeric proteins are much more abundant. The approximate sizes of the truncated products are consistent with protein cleavage at several distinct positions near the carboxy-terminal end of TAND.
One possible mechanism for Taf1p truncation in vivo is proteolysis at specific amino acid sequences between residues 86 and 196 of Taf1p. This possibility was tested by systematically substituting blocks of 5 amino acids in this region with the sequence Gly-Ser-Ala-Ala-Ala (GSAAA). GSAAA was substituted for 23 sites between residues 77 and 191 of d1y2 and y1d2 and the effect of these substitutions on truncation of chimeric Taf1p was examined (data not shown). The majority of the substitutions caused subtle changes in the relative intensity of Taf1p truncation products. However, substitution at four different sites each eliminated 1 of the 5 major truncation products. The substitutions that had the largest effects on truncation included or were adjacent to methionine residues. These observations raised the possibility that these methionine residues could be used to initiate mRNA translation, leading to the synthesis of amino-terminally truncated protein synthesis products. As this possibility is consistent with other available data, the following experiments were conducted to validate or refute this idea.
Substitution of methionine with alanine residues near the carboxy-terminal end of TAND
If Taf1p truncation products are generated by initiating translation at methionine residues near the carboxy-terminal end of TAND, then truncation should be reduced or eliminated when methionine residues are substituted with alanine. This prediction was tested by substituting one or several of 10 methionine residues between residues 82 and 249 with alanine and examining the effect on the truncation of chimeric d1y2 and y1d2 Taf1p (Fig. 2). The 10 substituted methionine residues were at positions 82, 83, 85, 107, 137, 139, 176, 184, 188 and 249 (Fig. 2); results for d1y2 are shown in Fig. 2A and results for y1d2 are shown in Fig. 2B.
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The results were very similar for chimeric y1d2 Taf1p (Fig. 2B), with the exception that the largest band generated by truncated y1d2 proteins appears to correspond electrophoretically to band 3 of d1y2 (Fig. 1A, lanes 15, 16), which is initiated at Met107 (Fig. 2A). Although the Met[82, 83, 85]Ala substitution significantly decreased the intensity of this band (Fig. 2B, lane 2), the results with the Met[107, 137, 139, 176, 184, 188]Ala mutant demonstrate that it is translated from Met107, as the accumulation of band 2 was greatly enhanced relative to the Met[137, 139, 176, 184, 188]Ala mutant (compare lanes 11, 12 in Fig. 2B). These observations suggest that Met[82, 83, 85] is required for efficient translation from Met107, at least in case of the y1d2 derivative.
As described above (Fig. 1A, lane 13), low levels of the truncated forms of y1y2 Taf1p are observed in yeast cells. The results shown in Fig. 2C suggest that these proteins are generated by the same mechanisms that generate truncated products from chimeric Taf1p with dTAND1 or dTAND2.
Redundant and/or independent roles of closely positioned methionines in producing truncated forms of Taf1p
Figure 2 shows that the Met[176, 184, 188]Ala substitution eliminates band 5, but either Met176 or Met[184, 188] are sufficient to produce nearly WT levels of band 5 (compare lanes 5, 6, 7 and 1, 9, 10 in Fig. 2A,B). This implies that Met176 and Met[184, 188] may be redundant rather than independent translational initiators. Similar results were also obtained with regard to the redundancy of Met 82, 83 and 85 (Fig. 3A) for truncating d1y2 by alternative translational initiation. The Met[82, 83, 85]Ala substitution in d1y2 eliminates band 2 and increases bands 3 and 5 (Fig. 2A, lane 2). When combined with Met[107]Ala, which eliminates band 3 of d1y2 (Fig. 3A, lane 2), Met[82, 83, 85]Ala eliminates band 2, but substitution of any 1 or 2 of Met[82, 83, 85] does not reduce the amount of band 2 (Fig. 3A, upper panel). This result supports the conclusion that Met82, 83 and 85 are functionally redundant for the truncation of d1y2. However, the results with y1d2 were much less clear, at least in part because a very low level of band 2 is generated (Fig. 3A, lower panel).
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Mutations that reduce expression of full-length Taf1p do not affect the accumulation of truncated derivatives
The results described above strongly suggested that the truncated Taf1p derivatives were produced by alternative selection of translational initiation sites. However, it remained possible that they were produced by sequence-specific proteolysis by a protease that cleaves to the carboxy-terminal side of methionines. This possibility was tested by introducing mutations that prevent or strongly reduce expression of Taf1p, and determining if the amount of truncated Taf1p is also strongly reduced or eliminated.
Three approaches were used to reduce expression of y1y2, d1y2 and y1d2 Taf1p (Fig. 4). First, the AUG for initiating translation of full-length Taf1p was replaced with UAC, which codes for tyrosine (Fig. 4, lanes 2, 6, 10). Second, a frameshift mutation was created by inserting one cytosine residue 3' to the initiation codon, which generated a short open reading frame encoding MRKAAGIHRQWIGLDRHSFRQHRLRGQTAAR (d1y2, lane 3) or MRKAAGIRQDQLGQRR (y1d2, lane 7 and WT, lane 11). Third, the UCC for serine at position 7 was replaced with a UAG stop codon (lanes 4, 8, 12). These mutations prevented expression of full-length d1y2 and y1d2 (compare Fig. 4 lanes 1 and 5 with lanes 2–4 and 6–8, respectively), but a significant amount of full-length y1y2 was still expressed when the initiation codon was replaced with UAC (lane 10). This result is difficult to explain, because there is a stop codon 9 bp upstream of and in frame with the substituted methionine and only ACG or CUG codons are normally used as alternative initiation codons in eukaryotes (Kozak 1999). The other two approaches (frameshift mutation or insertion of an in-frame stop codon) strongly reduced expression of full-length Taf1p y1y2 (lanes 11, 12).
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Figure 4 also shows the growth properties of strains expressing reduced quantities of y1d2, d1y2 and y1y2 Taf1p. As we observed neither additive growth defects nor recovery of TS phenotypes with these mutations (lanes 1–8, lower panel), the production of small amounts of full-length d1y2 and y1d2 proteins may not have any impact on cell growth. In addition, the low level of full-length WT Taf1p synthesized when the codon for Met1 is replaced by UAC does not support cell growth at 37 °C (lane 10). This mutation may lead to synthesis of a non-functional Taf1p or, alternatively, the amount of WT Taf1p may be insufficient to support growth at 37 °C.
The sequence encoding Drosophila TAND induces a downstream shift of transcriptional initiation sites
The results presented above do not explain why the presence of dTAND1 or dTAND2 influences selection of the translational initiation sites for Taf1p. However, it is possible that dTAND influences the core promoter binding properties of Taf1p in the TFIID complex, since Taf1p is known to be involved in the recognition of core promoter structures (Chalkley & Verrijzer 1999; Mencia & Struhl 2001; Oelgeschlager et al. 1996). If this is the case, it is predicted that novel transcription initiation sites might be utilized in downstream regions of the gene and short mRNAs with truncated open reading frames would be produced.
This idea was tested by sizing capped, full-length TAF1 mRNA using RLM-RACE (RNA Ligase Mediated Rapid Amplification of cDNA Ends) (Schaefer 1995). Total RNA was prepared from y1y2, d1y2 and y1d2 strains (Fig. 5A) and TAF1 mRNA was PCR amplified and sequenced. Figure 5 shows the deduced transcription initiation sites in TAF1 (Fig. 5A, lanes 1–3). The coordinates shown are relative to the A of the first AUG codon (Fig. 5B). The accuracy of this mapping was validated for the WT strain by primer extension analysis (data not shown). The results clearly show that TAF1 mRNA transcripts were initiated at downstream sites in TAF1 encoding d1y2 or y1d2 (Fig. 5A, compare lanes 1–3). In addition, transcripts encoding truncated y1d2 were shorter than transcripts encoding d1y2 derivatives, which agrees with the sizes of the truncated forms of y1d2 and d1y2 Taf1p (e.g. Figure 1B, lanes 2, 3). Some of the short transcripts are also produced from WT TAF1 (white arrows in lane 3, Fig. 5A), which is consistent with the evidence described above that similar truncation mechanisms apply to chimeric and WT Taf1p (Fig. 2C). Although the precise relationship between these short TAF1 transcripts and truncated protein products has not been determined, it is likely that they are related to the truncated forms of chimeric d1y2 and y1d2 Taf1p.
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Previous studies and Fig. 4 of this study demonstrate that d1y2 and y1d2 Taf1p do not support growth of yeast cells lacking WT Taf1p at 37 °C (Fig. 4, lanes 1, 5) (Kotani et al. 1998). This result suggests that the Drosophila subdomains are not functionally equivalent to yTAND subdomains. However, it is also possible that the chimeric Taf1p would be functional in yeast if expressed at a sufficiently high level to support growth at 37 °C. In addition, large amounts of truncated Taf1p might inhibit growth of yeast at 37 °C.
These possibilities were investigated by determining the growth phenotype of the Met-to-Ala substitution strains, which express variable amounts of full-length and/or truncated forms of chimeric or WT Taf1p (Fig. 2). The data indicate that some y1d2 but no d1y2 mutants grow at 37 °C (Fig. 6A,B). The following Met-to-Ala substitutions significantly improved growth: Met[82, 83, 85, 107, 137, 139, 176, 184, 188]Ala (Fig. 6B, lane 9), Met[82, 83, 85, 107, 137, 139, 176, 184, 188, 249]Ala (lane 10) and Met[107, 137, 139, 176, 184, 188]Ala (lane 14). However, in species with Met-to-Ala substitutions that resulted in the production of more truncation products than full-length protein (Fig. 2B, lanes 2–6 and 9–11), growth remained temperature sensitive at 37 °C (Fig. 6B, lanes 4–8, 11–13). The main conclusion of this experiment is that dTAND2 but not dTAND1 substitutes functionally for its Drosophila counterpart, but only if the full-length chimeric Taf1p is produced at a sufficiently high level or the expression ratio of truncated to full-length proteins is reduced in vivo.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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Recently, we identified a third TBP binding domain, TAND3, which is located between Met[82, 83, 85] and Met[137, 139] of Taf1p (Takahata et al. 2003). The observation that Taf1p lacking TAND(1 +2) and Taf1p lacking TAND(1 +2 +3) are functionally distinct also suggests that the in vivo mechanism that generates several truncated forms of Taf1p is physiologically relevant (Takahata et al. 2003). For example, these TAND-less Taf1p might regulate transcription of specific groups of genes. A small number of genes are significantly affected in a TAND strain (Chitikila et al. 2002) (K. Ohtsuki, K. Shirahige and T. Kokubo, unpublished observation), but it is not known which gene(s) is specifically regulated by TAND-less Taf1p. This question can now be addressed by expressing full-length Taf1p in the absence of truncated Taf1p by substituting 10 internally located methionine residues with alanine (e.g. see Fig. 2C, lane 8). Yeast cells carrying this Taf1p mutant produce little or no truncated forms of Taf1p and do not show any adverse effects on growth or other obvious phenotype (K. Kasahara, M. Kawaichi and T. Kokubo, unpublished observation). Thus, TAND-less Taf1p appears to be involved in the expression of a limited number of genes. Alternatively, it may regulate expression of different sets of genes under specific growth conditions.
This study also showed that dTAND2 substitutes functionally for yTAND2, but dTAND1 does not substitute for yTAND1 (Fig. 6). This result is consistent with earlier biochemical data indicating that dTAND1 binds stably to TBP, whereas yTAND1, yTAND2 and dTAND2 bind to TBP in the presence of other subdomains (Kokubo et al. 1998; Kotani et al. 1998). In addition, TAND2 is more highly conserved in different species than TAND1 (Kokubo et al. 1998; Nishikawa et al. 1997), suggesting that its biological role is also conserved through evolution. The previously proposed two-step handoff model for TFIID activation also predicts that TAND2 should be highly conserved, since the model suggests that TAND1 is a direct target for a wide variety of activators, but TAND2 is a target for TFIIA that is highly conserved among species (Bagby et al. 2000; Kotani et al. 2000).
Transcription of TAF1 encoding y1d2 or d1y2 Taf1p utilizes downstream transcription initiation sites (Fig. 5), which may precipitate the shift to downstream major translational initiation sites; however, the relationship between specific truncated TAF1 mRNAs and specific truncated protein species is not clear and multiple truncated protein species might be produced from a single truncated mRNA by ‘reinitiation’ or ‘context-dependent leaky scanning.’ These two processes involve use of downstream AUG codons and are exceptions to the ‘first AUG rule’ (reviewed in Kozak 2002). There is ample precedent for such exceptions as means to regulate the amount and/or the ratio of multiple isoforms of transcription factors in response to environmental stimuli (Kozak 2002).
The presence of multiple methionine clusters at the carboxy-terminal junction of TAND implies a process similar to ‘context-dependent leaky scanning’, in which downstream AUG codons are used to initiate translation when the ribosome encounters an AUG in an unfavourable context (Kozak 2002). Figure 4 shows that translational initiation at Met[82, 83, 85] increases dramatically when the first AUG codon of y1y2 is mutated (Fig. 4, lanes 9–12) and a novel pattern of truncated Taf1p isoforms results (lanes 10–12). According to the ‘leaky scanning’ mechanism, relative expression of multiple isoforms should not depend on the presence or absence of the first AUG codon. Furthermore, in case of the y1d2 TAF1 gene, downstream initiation does not increase even when the first AUG codon is disrupted (lanes 5–8). This strongly suggests that full-length Taf1p and truncated Taf1p are translated from different subpopulations of mRNA. However, the observation that Met[82, 83, 85] is required for the efficient translation from Met107 of the y1d2 derivative (Fig. 2) is consistent with translation of multiple isoforms from a single species of mRNA. Additional studies are needed to resolve this and other questions about the factors that influence truncation of Taf1p. For example, one methionine residue in dTAND1 is not used for translational initiation (Fig. 2), indicating that multiple factors determine which methionine residues are selected for initiating translation.
The Taf1p mRNAs encoding y1d2 and d1y2 are much more heterogeneous in size than the mRNAs encoding y1y2 (Fig. 5), suggesting that transcription initiation in TAF1 may be shifted downstream due to the presence of dTAND1 or dTAND2 (Fig. 5). Since very few capped mRNA species are detected that initiate in dTAND1 or dTAND2 (K. Kasahara, M. Kawaichi and T. Kokubo, unpublished data), it is unlikely that their latent strong promoter activities interfere with transcription from the native promoter. Although several studies show that Taf1p plays a role in the recognition of eukaryotic promoters (Chalkley & Verrijzer 1999; Mencia & Struhl 2001; Oelgeschlager et al. 1996), it is unclear how this might affect transcription from the core promoter. However, it appears that the expression of chimeric Taf1p may alter promoter selection by TFIID. Another possibility is that mRNAs encoding y1d2 and d1y2 are unstable, which might induce alternative promoters to support cell growth (Fig. 5). Nevertheless, it seems likely that selection of transcription initiation sites plays an important role in producing truncated forms of Taf1p. Consistent with this, recent studies have suggested that translation may occur in nuclei, which raises the possibility that transcription and translation could be more tightly coupled than previously envisioned (Brogna et al. 2002; Iborra et al. 2001).
In summary, this study demonstrates that truncated forms of WT and chimeric Taf1p are produced by an unusual mechanism involving alternative transcription and translation initiation sites in the TAF1 gene and mRNA, respectively. The specific biological role of truncated WT Taf1p remains to be determined in future experiments.
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Experimental procedures |
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Standard techniques were used for yeast growth and transformation (Lundblack 1998). Yeast strains used in this study are listed in Supplemental Table S1. All strains were generated from Y22.1 (taf1 strain) (Kokubo et al. 1998) using a plasmid shuffle technique.
Construction of recombinant plasmids encoding TAF1 genes
pM888, pM1007, pM985 and pM986 were constructed by inserting a DNA fragment encoding three repeats of the HA epitope tag at the amino-terminus of Taf1p encoded by pM11, pM10, pM756 and pM758 (Kotani et al. 1998), respectively. Similarly, pM1169, pM1001, pM978 and pM979 were constructed by inserting a DNA fragment encoding four repeats of the HA epitope tag at the carboxy-terminus of Taf1p encoded by pM11, pM10, pM756 and pM758, respectively.
pM1169 was subjected to site-specific mutagenesis (Kunkel et al. 1987) to create pM1689, pM1690, pM1691, pM1692, pM1693, pM1656, pM1694, pM1657 and pM1695 using oligonucleotides TK1308, TK1309, TK1310, TK1311, TK1312, TK1226, TK1313, TK1227 and TK1314, respectively. The oligonucleotides used in this study are listed in Supplemental Table S2. pM978 was mutagenized to create pM2302, pM1830, pM1831, pM1832, pM1833, pM1834, pM1835, pM1836, pM1837, pM2303, pM2304, pM2305, pM2306, pM2307, pM2308, pM2309, pM2310, pM2311, pM1783, pM1784, pM1785, pM1786 and pM1826 using oligonucleotides TK1526, TK1427, TK1428, TK1429, TK1430, TK1431, TK1432, TK1433, TK1434, TK1527, TK1528, TK1529, TK1530, TK1531, TK1532, TK1533, TK1534, TK1535, TK1315, TK1316, TK1317, TK1318 and TK1424, respectively. The same set of oligonucleotides were used for mutagenesis of pM979 to create pM2319, pM2320, pM2321, pM2322, pM2323, pM2324, pM2325, pM2326, pM2327, pM2328, pM2329, pM2330, pM2331, pM2332, pM2333, pM2334, pM2335, pM2336, pM1787, pM1788, pM1789, pM1790 and pM1827.
The methionine residues at aa positions 82, 83, 85, 107, 137, 139, 176, 184, 188 and 249 were changed one at a time to alanine by site-specific mutagenesis using oligonucleotides TK2139, TK2140, TK2141, TK1538, TK2037, TK2038, TK1540, TK1425, TK1426 and TK1641, respectively. Groups of 2 or 3 methionine residues were also changed to alanine. Methionine clusters including aa's [82, 83, 85], [82, 83], [82, 85], [83, 85], [137, 139] and [184, 188] were changed to alanine using oligonucleotides TK1536, TK2142, TK2143, TK2144, TK1539 and TK1438, respectively. The quadruple substitution, i.e. Met[82, 83, 85]Ala +Gly86Met, was constructed by site-specific mutagenesis using oligonucleotide TK1537. A frame shift mutation (AUG to AUGC) and two codon substitution mutations (AUG to UAC and UCC to UAG) were introduced by oligonucleotides TK1638, TK1637 and TK1639/TK1640, respectively. (Note that TK1640 was used for pM978, whereas TK1639 was used for pM1169 and pM979) Appropriate combinations of these changes were made to generate the series of plasmids from pM1169, pM978 and pM979 (Supplemental Table S1).
pM2374, encoding the y1d2 derivative of Taf1p carrying the Tyr129Ala substitution, was created from pM979 by site-specific mutagenesis using the oligonucleotide TK1653. The 5.3 kb NotI-PstI DNA fragments encoding the TAF1 genes of pM978, pM979 and pM2374 were subcloned into the NotI/PstI sites of pRS424 (Christianson et al. 1992) to create pM2713, pM2707 and pM2708, respectively.
pM2073 was constructed by inserting a DNA fragment encoding three repeats of the FLAG epitope tag at the carboxy-terminus of Taf1p encoded by pM11. The 3.2 kb and 2.6 kb SpeI-SpeI DNA fragments encoding WT and 2-188 amino acids TAF1 genes were amplified by PCR with primer pairs TK6414/TK18 and TK6416/TK18, respectively, using pM2073 as a template. These amplified DNA fragments were subsequently subcloned into the SpeI site of pM5049, which was generated from the p414-TEF expression vector (Mumberg et al. 1995; kindly provided by Dr Martin Funk) by transferring its 0.73 kb SacI-KpnI promoter-terminator cassette into the same sites of pRS425.
Immunoblot analysis
Immunoblot analysis was carried out as previously described (Kotani et al. 1998). Polyclonal antibodies against Taf1p were prepared as described (Kotani et al. 1998). A monoclonal antibody against the HA epitope was purchased from Santa Cruz Biotechnology, Inc.
Characterization of the transcriptional initiation site
The 5' ends of the TAF1 cDNAs encoding y1y2, d1y2 and y1d2 derivatives were mapped by RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) using the FirstChoiceTM RLM-RACE Kit (Ambion) with total RNA and TK3434 and TK3523 gene-specific primers. Total RNA was prepared from yTK2741 (y1y2), yTK2200 (d1y2) and yTK2205 (y1d2) strains using the RNeasy Midi Kit (Qiagen). DNA fragments were resolved by 5% polyacrylamide gel electrophoresis and cloned into pBluescript II (Stratagene). The nucleotide sequences of the 5' ends of cloned TAF1 cDNAs were determined.
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Supplementary material |
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Figure S1 (A) The effect of temperature on the shift of transcription initiation sites of the chimeric-TAND-containing TAF1 genes. RLM-RACE (RNA Ligase Mediated Rapid Amplification of cDNA Ends) was conducted for total RNA from strains expressing y1y2 (lanes 2, 5, 8), d1y2 (lanes 3, 6, 9) and y1d2 (lanes 4, 7, 10) that were harvested at 0 (lanes 2-4), 2 (lanes 5-7) and 24 (lanes 8-10) hours after a temperature shift from 30 to 37 °C. Amplified PCR products were resolved by 2% agarose gel electrophoresis and stained with ethidium bromide. The band profiles obtained for y1y2, d1y2 and y1d2 appeared to be the same for all temperature conditions. Thus we conclude that temperature does not affect the selection of transcriptional initiation sites of WT and chimeric TAF1 genes. (B) Immunoprecipitation of TFIID containing N-terminally truncated Taf1p derivatives. Whole cell extracts (WCE) were prepared from strains expressing a C-terminally HA-tagged Taf1p derivative lacking its N-terminal portion as indicated, as well as untagged WT Taf1p. Aliquots of the WCE were immunoprecipitated with anti-TAF11 polyclonal antibodies. Proteins co-precipitating with Taf11p were fractionated by SDS-PAGE, transferred to nitrocellulose membranes and probed with anti-HA monoclonal antibodies. An asterisk denotes a Taf1p derivative incorporated into TFIID.
Table S1 S. cerevisiae strains used in this study.
Table S2 Oligonucleotides used in this study.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: kokubo{at}tsurumi.yokohama-cu.ac.jp
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References |
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Received: 5 March 2004
Accepted: 1 June 2004
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