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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Curr Biol. Author manuscript; available in PMC 2011 May 11.
Published in final edited form as:
PMCID: PMC3033761
NIHMSID: NIHMS191742

Proteolytic instability and the action of non-classical transcriptional activators

Summary

Several transcriptional activators, called ‘classical’ because each bears a natural acidic activating region attached to a DNA binding domain, are proteolytically unstable in yeast, and it has been suggested that this instability is required for transcriptional activation [1-3]. Here we test the generality of that proposal by examining a set of activators (called ‘non-classical’) that lack activating regions. These activators (e.g. LexA-Gal11) comprise a LexA DNA binding domain fused to a component of the Mediator, and are believed to insert the latter into the Mediator and recruit it (and, indirectly, other components required for transcription) to a gene bearing LexA sites [4-8]. We find that three, and only three Mediator subunits, all from its tail domain, work as activators when fused to LexA. All three are unstable, and for the case analyzed in detail, stabilization decreases activity. Thus to the extent tested, both classical and non-classical activators work most efficiently when proteolytically unstable.

Results and Discussion

We constructed genes encoding fusion proteins of the form Med-LexA by integrating LexA DNA at the end of genes encoding 17 of 25 known Mediator subunits. Each hybrid gene was expressed from its native promoter at its ordinary chromosomal location. Table 1 shows that the transcriptional activating function of each fusion measured by β-galactosidase levels expressed from an integrated GAL1-LacZ reporter bearing LexA sites. Only three fusions elicited significant levels of β-galactosidase – those bearing, in addition to LexA, the Mediator subunits Gal11, Med2, and Pgd1. All three of these subunits are found in the tail domain of the Mediator.

Table 1
Activities of non-classical activators

Figure 1A shows that all three of the fusions that activate transcription − Gall1-, Med2-, and Pgd1-LexA – are unstable as visualized by their degradation following addition of cycloheximide. The figure also shows that, in contrast, fusions bearing either a subunit from the head domain (Srb2) or middle domain (Med1), which do not activate, are not unstable. We did not test the stabilities of other Med-LexA fusions. Figure 1B shows that whereas the native Gal11 protein is unstable, the native forms of Med2, Pgd1 and LexA are not. We chose Med2-LexA for further study.

Figure 1
Protein stabilities

As noted in the Summary, Mediator proteins fused to a DNA binding domain (DBD) activate transcription by integrating into the Mediator and recruiting the complex to the promoter by virtue of the attached DBD. This model was supported by the finding that LexA-Gal11 complemented the defect caused by deletion of WT GAL11 [4]. The experiment of Figure S1 extends this observation to the case of Med2-LexA. Thus, as shown in the figure, Med2-LexA corrects the growth defect caused by deletion of MED2 itself.

We considered the possibility that proteolysis of Med2-LexA occurred only as it activated transcription. The experiment of Figure 2 shows that this cannot be so. Thus, although the activity of Med2-lexA was strongly inhibited (squelched) by overexpression of either LexA or of Med2 (Figure 2A), in neither of those scenarios was the Med2-LexA rendered stable (Figure 2B and C). A further result of the squelching experiment can also be seen in Figure 2C: not only did the activity of Med2-LexA become significantly inhibited when Med2 was overexpressed, so too did the level of the fusion protein. Such an effect on the level of the fusion protein was not observed in cells overexpressing LexA (Figure 2B). The results suggest that a negative autoregulatory effect maintains the concentration of Med2 below some specified level (i.e., at high concentrations, Med2, directly or indirectly, turns off expression of its own gene.). Another possibility is that high concentrations of Med2 displace Med2-LexA from the Mediator and thereby, for some reason, render it more sensitive to proteolysis.

Figure 2Figure 2
Effects of overexpression of LexA and Med2

Figure 3A shows that Med2-LexA was stabilized in cells bearing temperature-sensitive (ts) pre1-1 pre4-1 double mutations of the 20S proteasome subunits and grown at the non-permissive temperature [9]. Figure 3B shows that the fusion was also stabilized by growth at the non-permissive temperature of a mutant strain bearing the cdc53-1 ts mutation [10]. This mutation inactivates, at the high temperature, the scaffold subunit cullin found in the SCF family of E3 ligases.

Figure 3Figure 3
The activity of a stabilized Med2-LexA

We introduced Med2-LexA, on a plasmid, into strains each of which is deleted for, or carries a temperature sensitive mutation in, one or another of 20 F-box proteins. These F-box proteins are known or predicted to be the specificity determinants of the SCF-type E3 ligases [2, 11]. In only one such strain, that bearing deletion of DIA2, was Med2-LexA found to be stable (not shown). When expressed from its native MED2 promoter, Med2-LexA was also stable in the dia2Δ strain (Figure 3C). Dia2 has not been found to be involved in degradation of any classical transcriptional activator. In this strain, Med2-LexA stimulated production of β-galactosidase from the reporter at least 5-fold less efficiently than it did in a WT strain. The level of LacZ mRNA was also decreased by the dia2 deletion, but the effect was less dramatic than the effect on the protein level (Figure 3D). In a control experiment, induction of GAL1 mRNA by Gal4 was identical in WT and dia2 deleted strains (not shown). Also not shown, the san1 mutation, which eliminates the protein quality control pathway in the nucleus [12], had no effect on the stability of Med2-LexA or of Pgd1-LexA, the two fusion proteins tested.

We undertook these experiments to challenge the notion that transcriptional activators must be proteolytically unstable to work with full efficiency. We reasoned that were the instability noted with classical activators such as Gal4 and Gcn4 some accidental attribute of their activating regions, then an entirely separate class of activators, that work in a different way, would, in some cases at least, work with full efficiency as stable proteins. But this expectation has been confounded. Of the three Mediator components that work as activators when attached to a DNA binding domain, one (Gal11) is inherently unstable, and the other two (Med2 and Pdg1) become unstable when attached to LexA. We do not know why these latter two fusions are unstable – it is possible that instability is some accidental effect of fusing these otherwise stable subunits to LexA. Nevertheless, the instability is required for full activity: where we eliminated a relevant F-box protein (Dia2) and rendered a fusion protein (Med2-LexA) stable, its activating activity was reduced. Thus our results are consistent with the idea that instability of transcriptional activators contributes to the efficiencies with which they work. Perhaps transcriptional complexes must continually turn over to sequentially bring to the promoter components required for steps leading and subsequent to initiation, and activator instability can help facilitate this turnover. A hint that this idea might be correct was the finding of Muratani et al. [2] that, stabilization of Gal4 (caused by deleting DSG1) has a much greater effect on translation of the mRNA read from the promoter than it does on mRNA production itself. They further showed that the Pol II recruited by Gal4 working in the dsg1 mutant was deficient in Ser5 phosphorylation of its CTD and in recruitment of the mRNA capping complex. We find here (see Figure 3D) that increasing the stability of Med2-LexA (by deleting DIA2) preferentially decreased the amount of translated protein compared to that of the mRNA elicited by the fusion activator.

An additional finding reported here is that amongst a wide array of Med-LexA fusion proteins tested, only those bearing one of three subunits from the Mediator tail domain work efficiently as activators. An array of previous reports have suggested that components of the tail domain (and in particular Gal11) are targets of transcriptional activating regions [4-8, 13-15]. The results, taken with those presented here, might reflect some stereospecific restrictions on how recruitment can be successfully effected, either by a classical or a non-classical, activator, to build a transcriptional complex.

Experimental Procedures

Yeast strains and plasmids

The parental Saccharomyces cerevisiae strain used in most of this study is JPY5 (MATα, ura3-52 his3Δ200 leu2Δ1 trp1Δ63 lys2Δ385), which was derived from the S288C background [4]. The cdc53-1 strain and its corresponding wild-type strain were derived from the W303 background [16]. The pre1-1 pre4-1 strain and its corresponding wild-type strain were obtained from Pengbo Zhou [17]. Strains bearing myc-tagged MED2 or PGD1 were constructed using a standard method [18], and a modified version of this method, which involves replacing myc with LexA, was used to construct strains expressing the Med-LexA fusion proteins. The med2Δ and dia2Δ strains were constructed by deleting its corresponding ORF using a one-step replacement method [18].

The reporter plasmid for integration carrying 2 lexA sites upstream of GAL1-LacZ was described previously [4]. A plasmid overexpressing LexA from the ACT1 promoter was also described previously [4], which was used to construct a plasmid overexpressing Med2-LexA by amplifying the MED2 gene from the yeast genomic DNA and then inserting it in frame 5′ to LexA.

Cell growth assays

Cells were grown overnight to mid-log phase in YPD media and washed and diluted with water to 0.1 at OD600. Then a series of 10x dilutions were made, 5ul of each dilution spotted on YPD (Glucose) and YPGal (Galactose) plates. Pictures were taken after cells were grown at 30°C for 3 days.

β-galactosidase assays

β-galactosidase activity was measured as described previously [19]. Each assay was performed at least in duplicate and the standard error was less than 20%. Relative activities are reported with average WT activity set to 1.

Cycloheximide Chase Assays

Protein degradation was assessed by cycloheximide chase assays as described in Muratani et al. [2]. Cells were grown in appropriate synthetic complete media containing 2% glucose overnight to mid-log phase. Then cycloheximde was added to the culture (50ug/ml final concentration), samples taken at the indicated time points, and steady state levels of protein of interest visualized by Western blotting using appropriate antibodies. The antibodies used were anti-LexA (ab14553 from Abcam), anti-Carboxypeptidase Y (CPY, A6428 from Molecular Probes), anti-Med2 (sc-28058 from Santa Cruz Biotechnology), anti-Myc (sc-40 from Santa Cruz Biotechnology), anti-Gal11 (generated in our lab). In cases where temperature sensitive mutants were involved, WT and ts cells were grown at the permissive temperature (25°C) overnight to mid-log phase, and then shifted to the restrictive temperature (37°C) for an hour before cycloheximide was added.

RT-PCR analysis

RNA was extracted, reverse transcribed, and quantified by RT-PCR as described previously [20]. For cDNA measurement, primers used are 5′- GCCGCTACAGTCAACAGCAA-3′ and 5′-ATATTCAGCCATGTGCCTTCTTC-3′ for LacZ cDNA, and 5′-CGTTCCAATTTACGCTGGTT-3′ and 5′- GGCCAAATCGATTCTCAAAA-3′ for ACT1 cDNA. LacZ mRNA levels were normalized to those of ACT1. Relative LacZ mRNA was reported with average WT mRNA level set to 1.

Supplementary Material

01

Acknowledgements

We thank Pengbo Zhou for yeast strains, Vidya Prabhu for oligos, and Gene Bryant, Monique Floer, Santosh Narayan and Vidya Prabhu for helpful discussions. This work is supported by National Institute of Health grants GM067728 to W.P.T. and GM32308 to M.P., Ludwig Professor of Molecular Biology.

Footnotes

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