ArgRII, a Component of the ArgR-Mcm1 Complex Involved in the Control of Arginine Metabolism in Saccharomyces cerevisiae, Is the Sensor of Arginine

Repression of arginine anabolic genes and induction of arginine catabolic genes are mediated by a three-component protein complex, interacting with specific DNA sequences in the presence of arginine. Although ArgRI and Mcm1, two MADS-box proteins, and ArgRII, a zinc cluster protein, contain putative DNA binding domains, alone they are unable to bind the arginine boxes in vitro. Using purified glutathione S-transferase fusion proteins, we demonstrate that ArgRI and ArgRII1-180 or Mcm1 and ArgRII1-180 are able to reconstitute an arginine-dependent binding activity in mobility shift analysis. Binding efficiency is enhanced when the three recombinant proteins are present simultaneously. At physiological concentration, the full-length ArgRII is required to fulfill its functions; however, when ArgRII is overexpressed, the first 180 amino acids are sufficient to interact with ArgRI, Mcm1, and arginine, leading to the formation of an ArgR-Mcm1-DNA complex. Several lines of evidence indicate that ArgRII is the sensor of the effector arginine and that the binding site of arginine would be the region downstream from the zinc cluster, sharing some identity with the arginine binding domain of bacterial arginine repressors

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

The Paf1 Complex Represses ARG1 Transcription in Saccharomyces cerevisiae by Promoting Histone Modifications ▿

The conserved multifunctional Paf1 complex is important for the proper transcription of numerous genes, and yet the exact mechanisms by which it controls gene expression remain unclear. While previous studies indicate that the Paf1 complex is a positive regulator of transcription, the repression of many genes also requires the Paf1 complex. In this study we used ARG1 as a model gene to study transcriptional repression by the Paf1 complex in Saccharomyces cerevisiae. We found that several members of the Paf1 complex contribute to ARG1 repression and that the complex localizes to the ARG1 promoter and coding region in repressing conditions, which is consistent with a direct repressive function. Furthermore, Paf1 complex-dependent histone modifications are enriched at the ARG1 locus in repressing conditions, and histone H3 lysine 4 methylation contributes to ARG1 repression. Consistent with previous reports, histone H2B monoubiquitylation, the mark upstream of histone H3 lysine 4 methylation, is also important for ARG1 repression. To begin to identify the mechanistic basis for Paf1 complex-mediated repression of ARG1, we focused on the Rtf1 subunit of the complex. Through an analysis of RTF1 mutations that abrogate known Rtf1 activities, we found that Rtf1 mediates ARG1 repression primarily by facilitating histone modifications. Other members of the Paf1 complex, such as Paf1, appear to repress ARG1 through additional mechanisms. Together, our results suggest that Rtf1-dependent histone H2B ubiquitylation and H3 K4 methylation repress ARG1 expression and that histone modifications normally associated with active transcription can occur at repressed loci and contribute to transcriptional repression

Complete DNA sequence of the yeast chromosome II

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms or are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of -110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.0SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe