The protein kinase Pho85 is required for asymmetric accumulation of the Ash1 protein in Saccharomyces cerevisiae.

The Ash1 protein is a daughter cell-specific repressor of HO gene transcription in Saccharomyces cerevisiae. Both ASH1 mRNA and protein are localized to the incipient daughter cell at the end of mitosis; Ash1 then inhibits HO transcription in the daughter cell after cytokinesis. Mother cells, in contrast, contain little or no Ash1 and thus are able to transcribe HO. We show that deletion of PHO85, which encodes a cyclin-dependent protein kinase, causes reduced transcription of HO and that this reduction is dependent on ASH1. In pho85 mutants, Ash1 protein is no longer asymmetrically localized and is present, instead, in both mother and daughter cells. Initially, it appears to be localized properly but then persists as daughter cells mature into mother cells. In contrast, ASH1 mRNA is localized appropriately to daughter cells in pho85 mutants. We observe that Ash1 protein is phosphorylated by Pho85 in vitro and that Ash1 stability increases in a pho85 mutant. These data suggest that phosphorylation of Ash1 by Pho85 governs stability of Ash1 protein.

Ash1p is a site-specific DNA-binding protein that actively represses transcription.

ASH1 encodes a protein that is localized specifically to the daughter cell nucleus, where it has been proposed to repress transcription of the HO gene. Using Ash1p purified from baculovirus-infected insect cells, we have shown that Ash1p binds specific DNA sequences in the HO promoter. DNase I protection analyses showed that Ash1p recognizes a consensus sequence, YTGAT. Mutation of this consensus abolishes Ash1p DNA binding in vitro. We have shown that Ash1p requires an intact zinc-binding domain in its C terminus for repression of HO in vivo and that this domain may be involved in DNA binding. A heterologous DNA-binding domain fused to an N-terminal segment of Ash1p functions as an active repressor of transcription. Our studies indicate that Ash1p is a DNA-binding protein of the GATA family with a separable transcriptional repression domain.

Transcriptional activity of transcription factor IIE is dependent on zinc binding.

The functions of individual basal transcription factors during the formation of an initiation complex by RNA polymerase II remain largely unknown. Transcription factor IIE (TFIIE) has recently been shown to bind to multiple targets in the initiation complex. To assess the role of zinc binding in basal transcription, we have mutated the predicted zinc-finger domain of human TFIIE. Atomic absorption spectroscopy using purified recombinant proteins revealed that the large subunit, TFIIE-56, is indeed a zinc-binding protein. Mutation of a cysteine residue in the putative zinc-finger domain abolished zinc binding. Moreover, mutant TFIIE-56 failed to support reconstituted basal transcription in vitro, suggesting that zinc binding is required for TFIIE function. However, gel-filtration experiments and protein affinity experiments suggest that mutant TFIIE-56 forms a stable heterotetramer with the small subunit, TFIIE-34, that is similar to wild type. Interestingly, gel mobility shift experiments reveal that loss of transcriptional activity by mutant TFIIE is correlated with its inability to stably assemble into the transcription complex. These findings establish that zinc binding by TFIIE may help form a specific structure that is required for stable entry into the transcription complex.

Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance.

The basal factor TFIIE is an important component of the RNA polymerase II transcription machinery. In our efforts to determine the role of TFIIE in the transcription process, we have begun to define the interactions between TFIIE and other basal transcription factors. Here we report that TFIIE binds selectively to the nonphosphorylated form of RNA polymerase II (IIa) and that this interaction is mediated by the 56-kD subunit of TFIIE. Additional binding studies reveal that TFII can interact with TBP as well as TFIID and that this interaction is mediated primarily via the 56-kD subunit. Our studies indicate that TFIIE also interacts with both subunits of TFIIF and with TFIIH, a multisubunit basal factor reported to catalyze RNA polymerase II CTD phosphorylation. Protein affinity assays demonstrate that TFIIE binds directly to ERCC-3, a DNA repair protein associated with TFIIH. More importantly, TFIIE affinity resin can selectively isolate transcriptionally competent TFIIH from a partially purified preparation and thereby may recruit TFIIH to the transcription complex in vivo. These multiple interactions between TFIIE, Pol II and TFIIH support a model in which TFIIE plays a role in promoter clearance as well as in the recruitment of proteins required for transcription-coupled DNA repair.

Structure and functional properties of human general transcription factor IIE.

The general transcription factor IIE (TFIIE) is an essential component of the eukaryotic RNA polymerase II initiation complex. We have isolated human complementary DNA clones for both the subunits of TFIIE. Using purified recombinant proteins we find that both subunits are essential to form a stable preinitiation complex and to reconstitute basal-level and Sp1-activated transcription in vitro. Analysis of their predicted amino-acid sequences reveals several intriguing structural motifs that could provide insight into the role of TFIIE in transcription initiation.

Structure-function studies on Escherichia coli MetR protein, a putative prokaryotic leucine zipper protein.

The Escherichia coli metR gene has been sequenced. The sequence predicts a protein of 317 amino acids and a calculated molecular weight of 35,628. This is about 15% larger than the protein from Salmonella typhimurium reported previously [Plamann, L.S. & Stauffer, G.V. (1987) J. Bacteriol. 169, 3932-3937]. The protein is a homodimer and contains a leucine zipper motif characteristic of many eukaryotic DNA-binding proteins. Replacement of two of the leucines in the leucine zipper region of the MetR protein, or substitution of proline for one of the leucines, results in loss of biological activity of the protein. In addition, truncation studies have identified a region on MetR that may be involved in the homocysteine activation of metE expression.

Methionine synthesis in Escherichia coli: effect of the MetR protein on metE and metH expression.

Studies by Urbanowski et al. [Urbanowski, M. L., Stauffer, L. T., Plamann, L. S. & Stauffer, G. V. (1987) J. Bacteriol. 169, 1391-1397] have identified a regulatory locus, called metR, required for the expression of the metE and metH genes. We recently purified the MetR protein from Escherichia coli and showed that it could stimulate the in vitro expression of the metE gene and autoregulate its own synthesis. In the present study, the purified MetR protein has been shown to stimulate the in vitro expression of the metH gene. Also, the in vitro synthesized MetE, MetH, and MetR proteins were shown to be biologically active. The transcription start sites for the metE and metR genes have been determined, and DNA footprinting experiments have identified regions in the metE-metR intergenic sequence that are protected by either the MetR or MetJ proteins.

Regulation of methionine synthesis in Escherichia coli: effect of the MetR protein on the expression of the metE and metR genes.

A plasmid (pRSE562) containing the metE and metR genes of Escherichia coli was used to study the expression of these genes and the role of the MetR protein in regulating metE expression. DNA sequence analysis of the 236-base-pair region separating these genes showed the presence of seven putative met boxes. When this plasmid was used to transform either wild-type E. coli, metE mutant, or metR mutant, MetE enzyme activity increased 5- to 7-fold over wild-type levels. The metR gene was subcloned from pRSE562, and this plasmid, pMRIII, relieved the methionine auxotrophy of a metR mutant after transformation. The metR gene was also cloned into a vector containing the lambda PL promoter, and the MetR protein was overexpressed and purified to near homogeneity. This protein, when added to an in vitro DNA-dependent protein synthesis system in which the MetE and/or MetR proteins were synthesized, caused a large increase in the expression of the metE gene but a decrease in the expression of the metR gene. The in vitro expression of both genes was inhibited by the MetJ protein and S-adenosylmethionine in the presence or absence of MetR protein. These results provide evidence that the product of the metR gene is a trans-activator of the expression of the metE gene and that the expression of the metR gene is under autogenous regulation and is repressed by the MetJ protein.