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.

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.

Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPBJ, were obtained by introducing a mutagenized RPBI plasmid into yeast cells, selecting for loss of the wild-type RPBI gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPBI mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPBJ 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 polymerases I, II, and III are highly conserved enzymes that are responsible for rRNA, premRNA, and small stable RNA synthesis, respectively (16,25). These enzymes are each composed of two very large subunits, which account for much of the molecular mass of the enzyme, and 8 to 11 smaller proteins. The two large subunits of RNA polymerase I are similar in sequence to the two large subunits of RNA polymerases II and III and to the two large subunits of the procaryotic enzyme (2,18,26). The two large subunits of the procaryotic enzyme bind DNA and nucleoside triphosphate substrates, contain the catalytic site for RNA synthesis, and interact with the transcription factor a (8,29). Sequence similarity between the two large procaryotic and eucaryotic subunits and evidence that the eucaryotic large subunits can bind to DNA and nucleoside substrates (7, 10) suggest that the eucaryotic homologs play similar roles.RNA polymerase II is highly conserved in subunit structure and sequence among eucaryotes (14,22,25). Comparison of the sequences of the largest RNA polymerase IL subunits from Saccharomyces cerevisiae (2), Caenorhabditis elegans (5), Drosophila inelanogaster (4, 15), and the mouse (1) reveal that almost 40% of the amino acid residues are invariant. The RNA polymerase II large subunits are also similar to their procaryotic counterparts. Much of the amino acid conservation between the large eucaryotic subunit and the procaryotic RNA polymerase 3' subunit occurs in multiple segments (1,2,4,5). Sequence similarities between the second largest subunits of eucaryotic and procaryotic RNA polymerases also occur in multiple segments (11,26). The presence of multiple segments of sequence similarity may reflect the fact that these large subunits have multiple functions. Invariant amino acid residues in and around these domains probably play essential structural and functional roles.We have begun a detailed genetic investigation of the two large RNA polymerase II subunit genes in S. cerevisiae. A systematic survey of mutations in the two large subunits has permitted the isolation of mutant cells that exhibit conditional and auxotrophic phenotypes. We have found that most of the amino acids altered by these mutations involve residues that are invariant among homologous subunits from * Corresponding author. a broad range of eucaryotes and discuss the implications of this observation her...