Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF.

Proteins interact with genomic DNA to bring the genome to life; and these interactions also define many functional features of the genome. SBF and MBF are sequence-specific transcription factors that activate gene expression during the G1/S transition of the cell cycle in yeast. SBF is a heterodimer of Swi4 and Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3). The related Swi4 and Mbp1 proteins are the DNA-binding components of the respective factors, and Swi6 mayhave a regulatory function. A small number of SBF and MBF target genes have been identified. Here we define the genomic binding sites of the SBF and MBF transcription factors in vivo, by using DNA microarrays. In addition to the previously characterized targets, we have identified about 200 new putative targets. Our results support the hypothesis that SBF activated genes are predominantly involved in budding, and in membrane and cell-wall biosynthesis, whereas DNA replication and repair are the dominant functions among MBF activated genes. The functional specialization of these factors may provide a mechanism for independent regulation of distinct molecular processes that normally occur in synchrony during the mitotic cell cycle.

Integrating functional genomic information into the Saccharomyces genome database.

The Saccharomyces Genome Database (SGD) stores and organizes information about the nearly 6200 genes in the yeast genome. The information is organized around the 'locus page' and directs users to the detailed information they seek. SGD is endeavoring to integrate the existing information about yeast genes with the large volume of data generated by functional analyses that are beginning to appear in the literature and on web sites. New features will include searches of systematic analyses and Gene Summary Paragraphs that succinctly review the literature for each gene. In addition to current information, such as gene product and phenotype descriptions, the new locus page will also describe a gene product's cellular process, function and localization using a controlled vocabulary developed in collaboration with two other model organism databases. We describe these developments in SGD through the newly reorganized locus page. The SGD is accessible via the WWW at http://genome-www.stanford.edu/Saccharomyces/

Using the Saccharomyces Genome Database (SGD) for analysis of protein similarities and structure.

The Saccharomyces Genome Database (SGD) collects and organizes information about the molecular biology and genetics of the yeast Saccharomyces cerevisiae. The latest protein structure and comparison tools available at SGD are presented here. With the completion of the yeast sequence and the Caenorhabditis elegans sequence soon to follow, comparison of proteins from complete eukaryotic proteomes will be an extremely powerful way to learn more about a particular protein's structure, its function, and its relationships with other proteins. SGD can be accessed through the World Wide Web at http://genome-www.stanford.edu/Saccharomyces/

RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals.

The large subunit of RNA polymerase II contains a highly conserved and essential heptapeptide repeat (Pro-Thr-Ser-Pro-Ser-Tyr-Ser) at its carboxy terminus. Saccharomyces cerevisiae cells are inviable if their RNA polymerase II large subunit genes encode fewer than 10 complete heptapeptide repeats; if they encode 10 to 12 complete repeats cells are temperature-sensitive and cold-sensitive, but 13 or more complete repeats will allow wild-type growth at all temperatures. Cells containing C-terminal domains (CTDs) of 10 to 12 complete repeats are also inositol auxotrophs. The phenotypes associated with these CTD mutations are not a consequence of an instability of the large subunit; rather, they seem to reflect a functional deficiency of the mutant enzyme. We show here that partial deletion mutations in RNA polymerase II CTD affect the ability of the enzyme to respond to signals from upstream activating sequences in a subset of promoters in yeast. The number of heptapeptide repeats required for maximal response to signals from these sequences differs from one upstream activating sequence to another. One of the upstream elements that is sensitive to truncations of the CTD is the 17-base-pair site bound by the GAL4 transactivating factor.

RNA polymerase II mutants defective in transcription of a subset of genes.

Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated. At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription. The transcriptional defects exhibited by one of these Ino- mutants (rpb2-2) were further investigated. The induction of GAL10 and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INO1 transcription. When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others. Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations. The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species.

Conditional mutations occur predominantly in highly conserved residues of RNA polymerase II subunits.

Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPB1, were obtained by introducing a mutagenized RPB1 plasmid into yeast cells, selecting for loss of the wild-type RPB1 gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPB1 mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPB1 and RPB2 homologs. These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues.

Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis.