MUTATIONS IN THE 2ND LARGEST SUBUNIT OF RNA-POLYMERASE-II CAUSE 6-AZAURACIL SENSITIVITY IN YEAST AND INCREASED TRANSCRIPTIONAL ARREST IN-VITRO

Yeast RNA polymerase II enzymes containing single amino acid substitut ions in the second largest subunit were analyzed in vitro for elongati on-related defects. Mutants were chosen for analysis based on their ab ility to render yeast cells sensitive to growth on medium containing 6 -azauracil. RNA polymerase II purified from three different 6-azauraci l-sensitive yeast strains displayed increased arrest at well character ized arrest sites in vitro. The extent of this defect did not correlat e with sensitivity to growth in the presence of 6-azauracil. The most severe effect resulted from mutation rpb2-10 (P1018S), which occurs in region H, a domain highly conserved between prokaryotic and eukaryoti c RNA polymerases that is associated with nucleotide binding. The aver age elongation rate of this mutant enzyme is also slower than wild typ e. We suggest that the slowed elongation rate and an increase in dwell time of elongating pol II leads to rpbB-10's arrest-prone phenotype. This mutant enzyme can respond to SII for transcriptional read-through and carry out SII-activated nascent RNA cleavage.