Phosphorylation of the Saccharomyces cerevisiae Snf1 kinase activation loop is determined by the integration of two reaction rates: the rate of phosphorylation by upstream kinases and the rate of dephosphorylation by Glc7. The activities of the Snf1-activating kinases do not appear to be glucose-regulated, since immune complex kinase assays with each of the three Snf1-activating kinases show similar levels of activity when prepared from cells grown in either high or low glucose. In contrast, the dephosphorylation of the Snf1 activation loop was strongly regulated by glucose. When de novo phosphorylation of Snf1 was inhibited, phosphorylation of the Snf1 activation loop was found to be stable in low glucose but rapidly lost upon the addition of glucose. A greater than 10-fold difference in the rates of Snf1 activation loop dephosphorylation was detected. However, the activity of the Glc7-Reg1 phosphatase may not itself be directly regulated by glucose, since the Glc7-Reg1 enzyme was active in low glucose toward another substrate, the transcription factor Mig1. Glucose-mediated regulation of Snf1 activation loop dephosphorylation is controlled by changes in the ability of the Snf1 activation loop to act as a substrate for Glc7.

Summary The five-subunit yeast Paf1 Complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.

Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1⌬ strains. We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the ␤ subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show that snf1⌬ strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1⌬ strains. Together, these findings indicate that the inositol auxotrophy of snf1⌬ strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.

The transcription factor LSF, identified as a HeLa protein that binds the simian virus 40 late promoter, recognizes direct repeats with a center-to-center spacing of 10 bp. The characterization of two human cDNAs, representing alternatively spliced mRNAs, provides insight into the unusual DNA-binding and oligomerization properties of LSF. The sequence of the full-length LSF is identical to that of the transcription factors eCP2 and LBP-lc and has similarity to the Drosophila transcription factor Elf-1/NTF-1. Using an epitope-counting method, we show that LSF binds DNA as a homodimer. LSF-ID, which is identical to LBP-ld, contains an in-frame internal deletion of 51 amino acids resulting from alternative mRNA splicing. Unlike LSF, LSF-ID did not bind LSF DNA-binding sites. Furthermore, LSF-ID did not affect the binding of LSF to DNA, suggesting that the two proteins do not interact. Of three short regions with a high degree of homology between LSF and Elf-1/NTF-1, LSF-ID lacks two, which are predicted to form j-strands. Double amino acid substitutions in each of these regions eliminated specific DNA-binding activity, similarly to the LSF-ID deletion. The dimerization potential of these mutants was measured both by the ability to inhibit the binding of LSF to DNA and by direct protein-protein interaction studies. Mutations in one homology region, but not the other, functionally eliminated dimerization.Regulation of the initiation of transcription in mammalian cells relies substantially on the activities of sequence-specific DNA-binding proteins. The functional analysis of cDNAs for transcription factors has therefore provided a means for understanding molecular mechanisms by which transcriptional activation and repression occur, both in vitro and in vivo. Such cDNA clones allow dissection of oligomerization, DNA-binding, and transcriptional activation activities.Transcription factors often bind DNA as oligomers, allowing recognition of repeated sequences in the DNA. Most such DNA-binding sites consist of inverted repeated sequences, which are readily recognized by symmetric dimeric or tetrameric proteins. On the other hand, proteins have developed a variety of strategies to bind direct repeats. The eukaryotic heat shock transcription factors are trimers, which allow recognition of both inverted and direct repeats with defined spacings (29,35). The phage transcriptional regulator XcII forms tetramers to interact with direct repeats, although the exact mechanism of interaction is not clear (17). Finally, dimeric proteins that bind direct repeats are relatively uncommon. As indicated above, when the dimerization interface involves the same region in each monomer, creating a twofold rotational axis of symmetry, the two DNA-binding domains in the dimer are positioned to recognize inverted repeats on DNA. In contrast, dimeric proteins that recognize direct repeats either must structurally separate such a twofold symmetric dimerization domain from the DNA-binding domains

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