INTRODUCTION

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MOT1 is an essential Saccharomyces cerevisiae protein implicated in the regulation of a diverse set of genes (8, 9, 15). MOT1 was originally identified by a mutation, mot1-1, that resulted in elevated levels of reporter gene expression driven by a weak promoter (9). Subsequently, MOT1 was uncovered in several other similar genetic screens (12, 13, 16, 19). In unrelated lines of work, MOT1 was identified as a TATA binding protein (TBP)-associated factor (TAF) (17) and as a protein with ATP-dependent TBP-DNA dissociating activity in vitro (2). Consistent with these biochemical activities, MOT1 can repress basal transcription in vitro (1, 2). The activity of MOT1 in vitro can also be at least partially overcome by factors such as TFIIA that bind to TBP and block the interaction of TBP with MOT1 and/or stabilize the binding of TBP to DNA (1). Additionally, the transcriptional activator GAL4-VP16 can overcome MOT1-mediated repression of basal transcription in vitro (2). These combined biochemical and genetic observations lead to the suggestion that MOT1 functions in vivo as a global repressor of basal transcription. This simple picture of MOT1 function was challenged, however, by the surprising observation that mutation of MOT1 can have no detectable effect on expression of many genes or actually lead to a decrease in gene expression of certain genes in vivo (8, 15, 19). The HIS4 gene is particularly interesting in this regard because, while in vitro experiments supported the model that MOT1 can repress HIS4 basal transcription (2), in vivo, MOT1 appears to activate HIS4 transcription (8, 15).

How can the known TBP-DNA dissociating activity of MOT1 in vitro be reconciled with its apparent function as an activator at some promoters in vivo? One possibility is that MOT1 affects HIS4 expression in vivo by an indirect mechanism. For instance, MOT1 might regulate the expression of a protein that itself regulates the expression of many genes. Alternatively, MOT1 might function as an activator of transcription by disassembling TBP (or TBP-containing complexes) bound to some promoters. Certain promoters might direct the assembly of stable but kinetically "dead" transcription complexes. If transcriptionally competent complexes can form on these promoters only at a low frequency, then MOT1-catalyzed TBP-DNA dissociation at these sites might increase the levels of steady-state mRNA by providing an opportunity for multiple attempts to form a competent preinitiation complex.

Another possibility is that MOT1 is targeted to TBP-DNA complexes formed on high-affinity sites that are not present in promoters. TBP binds with high affinity to a variety of AT-rich sequences (7, 11, 22), and it is likely that many spurious TBP-binding sites are fortuitously present in the genome. Binding of TBP to bona fide TATA boxes could then be stabilized from MOT1 action at promoters by the association of TBP with other general transcription factors (1). In this study, in vitro transcription experiments were performed to test the idea that MOT1 might function to redistribute TBP among promoter and nonpromoter sites. The results indicate that low levels of MOT1 (stoichiometric to TBP) function to activate basal transcription. In contrast, a molar excess of MOT1 which approximates the amount of MOT1 found in nuclear extracts from wild-type cells leads to repression of basal transcription. Furthermore, the behavior of MOT1 can be switched in vitro from that of a weak activator to that of a weak repressor by adjusting the amount of nonpromoter DNA present in the reaction mixture. These results support a model in which one function of MOT1 in vivo is to facilitate the distribution of a limiting pool of TBP between promoter and nonpromoter sites.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Nuclear extracts. Nuclear extracts were prepared from 4-liter cultures of either wild-type (JD194 [9]) or mot1-1 (JD215b [9]) yeast grown in yeast extract-peptone-dextrose (YPD) at 30°C. Cells were pelleted in 500-ml centrifuge bottles in a GS3 rotor spun at 4,000 rpm for 9 min in a Sorvall RC 5B centrifuge. All subsequent centrifugations were performed under these conditions unless otherwise noted. Cell pellets were then resuspended in a total volume of 270 ml of 50 mM Tris-HCl (pH 7.5) containing 30 mM dithiothreitol (DTT) and incubated at 30°C for 15 min with gentle shaking. Cells were then pelleted and resuspended in a total volume of 40 ml of YPD-S (10 g of yeast extract, 20 g of peptone, 20 g of glucose, and 182.2 g of sorbitol per liter) containing 60 mg of Zymolyase 100T (ICN), 2 µM pepstatin, 0.6 µM leupeptin, chymostatin (2 µg/ml), 1 mM phenylmethylsulfonyl fluoride, and 2 mM benzamidine. Cell suspensions were then incubated for up to 2 h at 30°C, and spheroplast formation was monitored by measuring the optical density at 600 nm of 10 µl of cell suspension in 1 ml of 1% sodium dodecyl sulfate and by visualization, under a light microscope, of spheroplast ghosts formed in 1% sodium dodecyl sulfate. The reaction was terminated by the addition of 540 ml of YPD-S when approximately 70 to 80% of the cells had been converted to spheroplasts. Cells were then pelleted and resuspended in 1 liter of YPD-S. Following a 30-min incubation at 30°C with gentle shaking, cells were again pelleted and washed twice with 540 ml of YPD-S, followed by one wash with 540 ml of 1 M sorbitol at 4°C. All subsequent steps were performed at 4°C. The cell pellets were resuspended in approximately 250 ml of buffer A (18% [wt/vol] polysucrose 400 [Sigma], 10 mM Tris-Cl [pH 7.5], 20 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 0.5 mM spermidine, 0.15 mM spermine, 3 mM DTT, and protease inhibitors as described above). Spheroplasts were then homogenized by two passes through a Yamada LH21 homogenizer at 1,000 rpm. Large cellular debris and unlysed cells were removed by five centrifugations in 250-ml bottles spun in a GSA rotor at 5,400 rpm for 5 min each. The nuclei were then pelleted by spinning the extract at 13,000 rpm for 30 min in an SS34 rotor, and the nuclei were then resuspended in 10 to 20 ml of buffer B (100 mM Tris-acetate [pH 7.9], 50 mM potassium acetate, 10 mM MgSO₄, 20% glycerol, 2 mM EDTA, 3 mM DTT, and protease inhibitors as described above) by using a Dounce homogenizer. To lyse the nuclei, 3 M ammonium sulfate (pH 7.6) was added to the nuclear suspension to achieve a final concentration of 0.5 M. Following stirring for 30 min, the debris was pelleted by spinning the lysate at 28,000 rpm for 75 min in an SW28 rotor. Ammonium sulfate (0.35 g/ml of nuclear extract) was then added, the extract was stirred for 30 min, and then nuclear proteins were pelleted by centrifugation at 20,000 rpm for 30 min in an SW28 rotor. Nuclear proteins were then resuspended in approximately 0.5 ml of buffer C (20 mM HEPES-KOH [pH 7.6], 10 mM MgSO₄, 10 mM EGTA, 20% glycerol, 5 mM DTT, and protease inhibitors as described above) and dialyzed against 1 liter of buffer C for 4 h. Protein concentrations were determined by Bradford assay using bovine serum albumin as a standard; protein concentrations were typically 30 to 40 mg/ml. The experiments described in this paper were performed with two independently prepared batches of nuclear extract from mot1-1 cells, and the results were indistinguishable.

Recombinant proteins. Recombinant yeast TBP was obtained from an Escherichia coli overexpression strain as previously described (21). MOT1 was obtained from a yeast overexpression strain and purified with antibody-coupled beads as previously described (3). One unit of MOT1 activity is defined as the amount of MOT1 required to completely dissociate all of the TBP-DNA complexes formed under the conditions previously described (1); MOT1 activity was assayed by gel mobility shift assay as previously described (1). Under these conditions, 1 U of MOT1 represents approximately 35 ng of MOT1 polypeptide, but it is important to note that a precise conversion between mass and activity is difficult since MOT1 specific activity is batch dependent and susceptible to degradation even in optimal storage buffers and as a result of freeze-thaw cycles.

In vitro transcription. In vitro transcription was performed essentially as described previously (2, 6), with plasmids containing the HIS4 (pSH387), CYC1 (52 TATA element; pCZGal3), or ACT1 (pSH385) core promoters (2). Transcription reactions were also performed with a plasmid (pTM04) containing a 542-bp fragment of the HIS4 gene obtained by PCR using the primers 5'-GGCTCGAGATTTGAGCAAGGAACTATTTTTGA-3' and 5'-CCGGATCCGGTCATTATTCAGAAAAAAAATTTTGT-3'. This fragment was cloned into the XhoI and BamHI sites of pSH387 to generate an in vitro transcription template which directs the synthesis of RNA, which can be quantitated, and whose ends can be mapped by using the same primer as was used for the other templates. All of the transcription reactions except those shown in Fig. 3 (lanes 8 to 14) were performed in buffer containing 10 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate, 10 mM magnesium acetate, 5 mM EGTA, and 3.5% glycerol. The reactions in Fig. 3 (lanes 8 to 14) were performed in buffer containing 4 mM Tris-acetate (pH 8), 60 mM potassium acetate, 5 mM magnesium acetate, and 4% glycerol; for unknown reasons, under these conditions MOT1's ability to bind to TBP-DNA is similar to that seen in glutamate-containing buffer but its ATP-dependent TBP-DNA dissociating activity is much greater than that detected in glutamate-containing buffer (not shown). Transcription reaction mixtures contained approximately 150 µg of nuclear extract protein, and specifically initiated RNA was detected by primer extension as previously described (20). The oligonucleotide duplexes used in Fig. 6 were obtained by combining 5'-CCCCGAC CGGGTGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCG-3' and 5'-CGCGCCCCCACCCCCTTTTATAGCCCCCCTTCAGGAACACCCGGTCGGGG-3' to obtain a wild-type TATA-containing DNA or by combining 5'-CCCCGACCGGGTGTTCCTGAAGGGGGGCTGTAAAAGGGGGTGG GGGCGCG-3' and 5'-CGCGCCCCCACCCCCTTTTACAGCCCCCCTTCAGGAACACCCGGTCGGGG-3' to obtain a DNA duplex containing the sequence TGTAAAAG in the TATA box. The DNAs were mixed in a solution containing 10 mM Tris-Cl (pH 8), 1 mM EDTA, and 0.1 M NaCl; boiled; and then slowly cooled to room temperature for 30 min before use.

Western blot analysis. Nuclear extract protein (10 µg) or recombinant MOT1 or TBP was fractionated on 10% protein gels and transferred to Immobilon P membranes (Millipore). The blots were probed with rabbit polyclonal anti-TBP antiserum (21) or rabbit polyclonal anti-MOT1 antiserum. The MOT1 antiserum was raised by using a bacterially expressed fragment of MOT1 encoding the C-terminal 67 amino acids. Immunoreactive bands were detected by enhanced chemiluminescence (Amersham ECL+), and because the relationship between band intensity and amount of protein is nonlinear, for purposes of quantitation, only bands of similar intensity were compared.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Quantitation of MOT1 in wild-type and mot1-1 nuclear extracts. The amount of MOT1 in nuclear extracts from wild-type and mot1-1 cells was determined by Western blotting using rabbit polyclonal antiserum raised against the C-terminal 67 amino acids of MOT1. Previous results showed that this C-terminal tail of MOT1 is essential for function both in vitro and in vivo (3). Consequently, any MOT1 molecules lacking an intact C terminus which are not detectable with this antiserum are inactive. As shown in Fig. 1A, MOT1 antiserum specifically detects a protein in wild-type nuclear extracts migrating with an apparent molecular mass of 175 kDa. This polypeptide comigrates with MOT1 purified from a yeast overexpression strain (yMOT1) as well as MOT1 purified from a baculovirus overexpression system (rMOT1) (Fig. 1A, right-hand panel). Both purified preparations of MOT1 dissociate TBP-DNA complexes in an ATP-dependent manner, and both of these preparations have similar specific activities (not shown; see Materials and Methods). The immune antiserum also detects a series of smaller proteins in yeast nuclear extract, which may be degraded forms of MOT1. Since an intact N terminus was also shown to be essential for MOT1's activity both in vitro and in vivo (3), N-terminally degraded forms of MOT1 are all presumed to be inactive. Extract from mot1-1 cells contains no detectable full-length MOT1 protein, although similar levels of smaller immunoreactive species are present in both wild-type and mot1-1 extracts (Fig. 1A, right-hand panel, lanes 1 and 2; Figure 1C, lanes 1 to 3). The amount of MOT1 in wild-type nuclear extract was estimated by comparing the signal obtained in nuclear extract with that from various amounts of purified baculovirus-expressed MOT1 (Fig. 1B). Based on this and other blots (not shown), there is approximately 90 ng of full-length MOT1 polypeptide in 10 µg of wild-type nuclear extract. Quantitation of TBP in these same nuclear extracts indicates that there is approximately 0.5 ng of TBP per 10 µg of nuclear extract protein (Fig. 1C). Additionally, the amount of TBP polypeptide is unchanged by the mot1-1 mutation. Remarkably, this indicates that MOT1 is present in vast excess (>20-fold molar excess) over TBP in our nuclear extracts.

View larger version (44K): [in this window] [in a new window]   FIG. 1.   Western blot analysis of wild-type and mot1-1 nuclear extracts. (A) Specificity of antiserum and comparison of native and recombinant MOT1. The two panels were loaded with identical protein samples; the left-hand panel was probed with preimmune serum, and the right-hand panel was probed with anti-MOT1 antiserum. Lanes 1 and 2 contain 10 µg of nuclear extract from wild-type and mot1-1 yeast cells, respectively. Lane 4 contains 250 ng of rMOT1; lane 3 contains a comparable amount of yMOT1 whose activity was not determined. The arrow indicates the position of full-length immunoreactive MOT1. (B) Quantitation of MOT1 in wild-type nuclear extract. Lane 1 contains 10 µg of total nuclear extract protein. Lanes 2 to 6 contain 90, 120, 210, 240, or 300 ng, respectively, of recombinant MOT1. The blot was probed as described for panel A, with anti-MOT1 antiserum. (C) Quantitation of MOT1 and TBP in wild-type or mot1-1 nuclear extract. Lane 1 contains 20 µg of wild-type nuclear extract protein. Lanes 2 and 3 contain 20 µg of total protein from two independently prepared samples of mot1-1 nuclear extract. Lanes 4 to 11 contain MOT1 purified from a yeast overexpression strain (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, or 5.0 U, respectively). Lanes 12 to 16 contain 1, 3, 9, 15, or 20 ng, respectively, of recombinant yeast TBP. The blot was probed with anti-MOT1 (top half) or anti-TBP (bottom half) rabbit polyclonal antiserum.

Activation and repression of basal transcription by MOT1 in vitro. To determine the effects of exogenously added MOT1 on basal transcription, increasing amounts of yMOT1 were added to transcription reaction mixtures containing a yeast promoter and mot1-1 nuclear extract. Specifically initiated RNAs were quantitated by primer extension as described elsewhere (20). Results obtained with the HIS4 and CYC1 promoters are shown in Fig. 2A, and quantitation of the band intensities obtained with a PhosphorImager are shown in Fig. 2B. The HIS4 promoter is of particular interest, since MOT1 was shown to repress HIS4 basal transcription in vitro but activate HIS4 transcription in vivo (2, 8, 15). Basal transcription is almost undetectable in wild-type nuclear extract (2), but the levels of basal transcription obtained with mot1-1 nuclear extract are readily detected (Fig. 2A, lanes 1 and 6) (2). The addition of low levels of MOT1 (1 to 2 U) results in weak activation of transcription. Since these reaction mixtures contain approximately 7.5 ng of TBP, this amount of MOT1 would be insufficient to disrupt all of the TBP-DNA complexes formed under our standard gel mobility shift or footprinting conditions with this level of purified TBP. When 8 U of MOT1 are added to an otherwise identical reaction mixture, basal transcription is weakly repressed (Fig. 2A, lanes 5 and 10). In our standard assays, using purified components, for MOT1's ATP-dependent TBP-DNA dissociating activity, 8 U of activity is just sufficient to disrupt all of the TBP-DNA complexes formed in a reaction mixture containing 7.5 ng TBP (not shown). Multiple replicates of the experiment shown in Fig. 2A established that the weak activation seen with low levels of yMOT1 is reproducible and statistically significant (Fig. 2C). This activation depends on the presence of the MOT1 polypeptide in the yMOT1 preparation and depends on a catalytically active MOT1 ATPase (Fig. 2C). The activity of ATPase-defective MOT1 is described more fully below.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Purified MOT1 can weakly activate or repress basal transcription in vitro. (A) MOT1 purified from a yeast overexpression strain was titrated into transcription reaction mixtures containing nuclear extract from mot1-1 cells and either the HIS4 core promoter or the CYC1 core promoter as indicated. The amount of MOT1 added is expressed as units of activity, with 1 U being the amount of MOT1 which can completely dissociate all of the TBP-DNA complexes formed under the conditions previously described (1). No MOT1 was added to the reaction mixtures in lanes 1 and 6; the amounts of MOT1 added to the other reaction mixtures were 1 U (lanes 2 and 7), 2 U (lanes 3 and 8), 4 U (lanes 4 and 9), and 8 U (lanes 5 and 10). Transcripts were detected by primer extension. The major start sites of transcription are identical to those previously observed both in vitro and in vivo (2). The bands that were quantitated are indicated by the arrows. (B) The relative amounts of specifically initiated RNA obtained in panel A are plotted versus the amount of MOT1 added. (C) Statistical analysis of the effect of exogenously added MOT1. The maximum stimulation of basal transcription by MOT1 is compared to the effects of adding mock-affinity-purified eluate obtained with whole-cell extract from cells containing MOT1 without an epitope tag (mock) or an equivalent amount of purified MOT1 harboring a point mutation which destroys ATPase activity (K1303A). Relative transcription levels are normalized to those in reactions which contained no additional eluate (control). The peak of activation by MOT1 varied slightly from experiment to experiment and also varied with different preparations of yMOT1, but the maximal activation was generally seen with 1 to 4 U of yMOT1 (see text). The error bars represent the standard deviation obtained by averaging the results of at least three independent experiments.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Recombinant MOT1 can activate or repress basal transcription driven by the HIS4 core promoter. (A) Experiments were performed as described in the legend to Fig. 2, but with increasing amounts of baculovirus-expressed MOT1 (rMOT1). Transcription reactions were performed in standard potassium glutamate-containing buffer (lanes 1 to 7) or in buffer containing potassium acetate (lanes 8 to 14) (see Materials and Methods). The reaction mixtures contained 10 ng (lanes 2 and 9), 50 ng (lanes 3 and 10), 100 ng (lanes 4 and 11), 300 ng (lanes 5 and 12), 600 ng (lanes 6 and 13), or 1,000 ng (lanes 7 and 14) of MOT1. The bands that were quantitated are indicated by the arrow. (B) Quantitation by phosphorimager analysis of the data shown in panel A. The graph on the left is of data from lanes 1 to 7, and the graph on the right is of data obtained from lanes 8 to 14. For comparison with the results obtained with purified MOT1 from yeast (yMOT1), the x axes also indicate the approximate units of MOT1 activity added.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   ATPase-defective MOT1 represses basal transcription. (A) Basal transcription reactions were performed as described in the legends to Fig. 2 and 3 except that reaction mixtures contained 10 ng (lane 2), 30 ng (lane 3), 100 ng (lane 4), 300 ng (lane 5), or 1,000 ng (lane 6) of MOT1 K1303A. The reaction mixture in lane 1 contained no added MOT1 K1303A. The bands that were quantitated are indicated by the arrows. (B) Quantitation of the data shown in panel A.

View larger version (35K): [in this window] [in a new window]   FIG. 5.   Recombinant MOT1 can activate or repress basal transcription driven by either the CYC1 or ACT1 promoters. (A) Basal transcription reactions were performed as described in the legends to Fig. 2 and 3, with 10 ng (lanes 2 and 9), 50 ng (lanes 3 and 10), 100 ng (lanes 4 and 11), 300 ng (lanes 5 and 12), 600 ng (lanes 6 and 13), or 1,000 ng (lanes 7 and 14) of MOT1. (B) Quantitation by phosphoimager analysis of the data shown in panel A.

Roles of MOT1, TBP, and GAL4-VP16 in overcoming repression by nonpromoter DNA. To test the idea that the effect of MOT1 on basal transcription depends on competition for TBP binding between different TBP binding sites, we first tested the effect of exogenously added nonpromoter DNA on basal transcription driven by the HIS4 promoter. In these experiments, increasing amounts of plasmid DNA (lacking a yeast promoter) were added to basal transcription reaction mixtures otherwise carried out as described above. As shown in Fig. 6A, addition of nonpromoter DNA led to a three- to fivefold decrease in basal transcription. This decrease appears to be due to the binding of TBP (or a TBP-containing complex) to nonpromoter sites on the plasmid, because the addition of increasing amounts of TBP rescues HIS4 basal transcription in reaction mixtures containing nonpromoter DNA (Fig. 6B, lanes 9 to 14). The idea that repression of basal transcription is due to binding of TBP to nonpromoter DNA is also supported by the observation that basal transcription can be repressed by addition of a TATA box-containing oligonucleotide but not by addition of an equimolar amount of an oligonucleotide duplex containing a single point mutation in the TATA box which prevents TBP binding (Fig. 6C). Basal transcription driven by the HIS4 core promoter could also be rescued by the addition of yMOT1 (Fig. 6B, lanes 2 to 8), suggesting that under these conditions, MOT1 might activate transcription by facilitating the distribution of TBP between promoter and nonpromoter sites. As an aside, the effects of the transcriptional activator GAL4-VP16 were compared under conditions under which excess nonpromoter DNA was present or absent. The results in Fig. 6D demonstrate that GAL4-VP16 activates HIS4 basal transcription fivefold in the absence of exogenous nonpromoter DNA and fourfold in the presence of nonpromoter DNA. Since the fold activation is similar but the activator does not reconstitute transcription to similar levels under these two conditions, we conclude that the activation seen here does not reflect recruitment of TBP (or TFIID) to the HIS4 promoter. In contrast, GAL4-VP16 activates transcription from the HIS4 basal promoter to similar levels in extracts from wild-type or mot1-1 cells (2), consistent with the idea that this activator functions in at least two steps in the process of preinitiation complex formation.

View larger version (44K): [in this window] [in a new window]   FIG. 6.   Repression of HIS4 basal transcription by nonpromoter DNA and effects of addition of TBP or GAL4-VP16. (A) Transcription reactions were performed with nuclear extract from mot1-1 cells. The reaction mixtures contained 0 µg (lane 1), 0.24 µg (lane 2), 1.2 µg (lane 3), or 2.4 µg (lane 4) of pKS II+ plasmid in addition to 0.25 µg of HIS4-containing plasmid template (see Materials and Methods). (B) Transcription reaction mixtures contained either 0 µg (lane 1) or 0.8 µg (lanes 2 to 14) of pKS II+. MOT1 purified from yeast was added to lanes 3 to 7 (0.5, 1.25, 2.5, 5, or 7.5 U, respectively); lane 8 contains a volume of mock-purified MOT1 equivalent to the volume of MOT1 added to lane 7. In addition to the TBP already present in the extract, recombinant yeast TBP was added to reaction mixtures in lanes 10 to 14 (1, 3, 10, 20, or 30 ng, respectively). The minus symbols in other lanes indicate that reaction mixtures contained TBP present in the nuclear extract but no additional recombinant TBP was added to the reaction. (C) HIS4 core promoter activity was assayed in the absence (lane 1) or presence of 50-mer oligonucleotide duplexes containing a wild-type TATA sequence (lanes 2 to 5) or a mutant TATA sequence, TGTAAAAG, which does not bind TBP (lanes 6 to 9). The reaction mixtures contained 3 ng (lanes 2 and 6), 10 ng (lanes 3 and 7), 30 ng (lanes 4 and 8), or 100 ng (lanes 5 and 9) of the indicated competitor oligonucleotides. (D) Transcription reaction mixtures contained pKS II+ (lanes 2 and 3) and/or GAL4-VP16 which was preincubated with DNA prior to the addition of mot1-1 nuclear extract. GAL4-VP16 activated transcription fivefold in the absence of pKS II+ and fourfold in the presence of pKS II+.

Differential effect of MOT1 on HIS4 basal transcription in reactions with different amounts of nonpromoter DNA. The observation that MOT1 can rescue basal transcription under conditions of excess nonpromoter DNA suggests that an equivalent amount of MOT1 has different effects on transcription, depending on the amount of nonpromoter DNA present in the reaction mixture. To test this, MOT1 was added to transcription reaction mixtures which contained or did not contain excess nonpromoter DNA. As shown in Fig. 7, an amount of MOT1 that leads to weak repression of transcription under standard conditions (Fig. 7A lanes 1, 2, and 5 to 7) caused weak activation when additional nonpromoter DNA was present in the reaction mixture (Fig. 7A lanes 3, 4, and 8 to 10). Another example of these differential effects is shown in Fig. 7B in which low levels of MOT1 activate basal transcription whether or not excess nonpromoter DNA is present (lanes 1, 2, 4, and 5), whereas increasing the concentration of MOT1 leads to either weak repression of transcription (lanes 1 and 3) or weak activation of transcription (lanes 4 and 6), depending on the amount of nonpromoter DNA which is present in the reaction mixture.

View larger version (26K): [in this window] [in a new window]   FIG. 7.   Comparison of the effects of MOT1 on HIS4 basal transcription in the presence and absence of nonpromoter DNA. Transcription reaction mixtures contained 3.4 µg of pKS II+ nonpromoter DNA as indicated and/or MOT1. (A) Transcription reactions were performed with the HIS4 core promoter with (+) or without () MOT1 purified from yeast. The reaction mixtures in lanes 2 and 3 contained 3 U of MOT1, those in lanes 6 and 9 contained 2 U of MOT1, and those in lanes 7 and 10 contained 8 U of MOT1. (B) Reactions were performed as described for panel A but with 250 ng (lanes 2 and 5) or 800 ng (lanes 3 and 6) of recombinant MOT1 from baculovirus.

View larger version (30K): [in this window] [in a new window]   FIG. 8.   Transcription driven by a larger (542-bp) fragment of the HIS4 promoter is repressed but not activated by MOT1. (A) Transcription reactions were performed with the plasmid containing the larger 542-bp fragment of the HIS4 promoter (lanes 1 to 9) or the HIS4 core promoter (lanes 10 to 12). The reaction mixtures in lanes 1 and 7 to 12 contained no added MOT1, whereas rMOT1 was added to the reaction mixtures in lane 2 (10 ng), lane 3 (30 ng), lane 4 (100 ng), lane 5 (300 ng), and lane 6 (1,000 ng). The reaction mixtures in lanes 8 and 11 contained 1.2 µg of pKS II+ nonpromoter DNA, and the reaction mixtures in lanes 9 and 12 contained 2.6 µg pKS II+. The bands that were quantitated are indicated by the arrow. (B) Quantitation of the data shown in panel A, lanes 1 to 6.