Mutations in the Saccharomyces cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracyl

RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of twelve polypeptides where Rpb1 is the largest subunit. In order to further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide depleting drug 6-azauracyl (6AU). In this work we have re-isolated the rpo21-24/rpb1-E1230K allele which reduces the interaction of RNAPII with TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the "lid" and "rudder," two small loops suggested by structural studies to play a central role in the separation of the RNA-DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of those two last mutants with soh1, spt4 and dst1 suggest differential in vivo effects.

Key Words: 6-azauracyl, RNA elongation, RNA polymerase II, RPB1, lid and rudder

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