Transcriptional activating regions target a cyclin-dependent kinase

Abstract

Several yeast activators are phosphorylated by SRB10, a cyclindependent kinase associated with the transcriptional machinery. Sites of phosphorylation are found outside the activating region in each case, and the modification has different physiological consequences in different cases. We show here that certain acidic transcriptional activating regions contact SRB10 as assayed both in vivo and in vitro. The interaction evidently positions each activator, as it activates transcription, so that it gets phosphorylated by SRB10, and thus a common mechanism targets disparate substrates to the kinase. S everal yeast transcriptional activators become phosphorylated as they activate transcription (1-3). Phosphorylation has various consequences: it affects nuclear export of Msn2, degradation of Gcn4, inducer-dependent activation by Gal4, and the activity of Sip4 (1-3). In each case, the activator is phosphorylated on residues positioned outside its activating and DNA binding regions. For example, the most important sites phosphorylated in Gal4 (S690, S696, and S699) lie in the region between the DNA binding domain and the principal activating region (2). Nevertheless, under physiological conditions, both the DNA binding and the activating regions are required for these phosphorylations, just as they are required for activity of the activator (2, 4).The kinase believed responsible for the phosphorylations described above is the cyclin-dependent kinase SRB10 complexed with the cyclin SRB11 (1-3). The SRB10͞11 pair is part of a larger complex that includes SRB8 and SRB9, and that complex can, by some reports, associate with the RNA polymerase II mediator (5, 6). SRB10͞11 is also believed to phosphorylate other substrates in the transcriptional machinery (5-7). How the various transcriptional activators are targeted to the SRB10 kinase has heretofore been unknown. Here, we show that the activating region of Gal4 contacts SRB10 as measured in binding assays performed in vitro and in vivo. Binding experiments performed in vitro indicate that other acidic activating regions also interact with SRB10. Materials and MethodsProteins. Purifications of recombinant CDK͞cyclins were performed as described (6,8). Yeast RNA polymerase II holoenzyme was purified as in ref. 9. GST-Gal4wt (840-874) and GST-Gal4mut (840-874) (which contains the mutations V864E and L868V) were purified as in ref. 10. TATA binding protein (TBP), Gal80, and other Gal4 derivatives were purified as described (10, 11).For Fig. 1B, Gal4 derivative bearing the F856C substitution was overexpressed in BL21 (DE3) pLysS bacterial strain. Cells were lysed in buffer A (20 mM Hepes, pH 7.5͞150 mM KCl͞ 0.1% Nonidet P-40͞20 M ZnCl 2 ͞3 mM DTT͞1 mM ␤-mercaptoethanol͞10% glycerol͞1 mM PMSF͞0.5 mM benzamidine) containing 6 M urea. After centrifugation (15 min at 10,000 ϫ g), the protein was purified by using a SP-Sepharose fast flow (Amersham Pharmacia) column under denaturing conditions. The relevant fractions were concentrated and refolded by rapid dilution into buffer A with...

Structure of the Mediator Subunit Cyclin C and its Implications for CDK8 Function

“…18 CDK8 binds the Gal4 activation domain. 19 CDK8 promotes ATP-dependent dissociation of preinitiation complexes, resulting in a positive effect on transcription. 20 CDK8/ cyclin C phosphorylates subunits of the general transcription factor TFIID, 20 and the Mediator subunit MED2.…”

Promoter-dependent Roles for the Srb10 Cyclin-dependent Kinase and the Hda1 Deacetylase in Tup1-mediated Repression inSaccharomyces cerevisiae

“…We have applied the same techniques that we used to analyze the tup1⌬ microarrays to determine the sets of genes derepressed when the functions of Srb10 and Hda1 are disrupted. We constructed an isogenic set of strains deleted for TUP1 or HDA1 or containing a mutant of Srb10 that lacks kinase activity (srb10 D304 ) (Liao et al, 1995;Ansari et al, 2002). We also made a strain lacking both Hda1 and Srb10 functions to determine the combined effects of the two mutations on expression levels.…”

Transcriptional activating regions target attached substrates to a cyclin-dependent kinase

“…In a previous article, we showed that each of an array of transcriptional acidic activating regions (yeast, mammalian, and artificial) binds Srb10 (5). The interaction, detected in vivo, was also observed in vitro with purified Srb10 alone (but not with Srb11 alone), with Srb10͞11, and with Srb10͞11 as part of the mediator complex (5).…”

“…Gst-fused proteins were purified as described (5). Gst-fusion proteins were expressed in BL21 (DE3) pLysS bacterial strain.…”

“…The effect of the modification is unique to each case. For example, phosphorylation of Gal4 increases the efficiency of induction of the galactose-responsive genes; that of Gcn4 and Ste12 leads to their proteolysis; that of Msn2 leads to its export from the nucleus; and that of Sip4 increases the efficiency of induction of glucoseresponsive genes (1)(2)(3)(4)(5). Srb10͞11 also phosphorylates a heptapeptide (YSPTSPS), 26 tandem copies of which comprise the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (6)(7)(8)(9).…”