Abstract
Yeast RNA polymerase II enzymes containing single amino acid substitutions in the second largest subunit were analyzed in vitro for elongation-related defects. Mutants were chosen for analysis based on their ability to render yeast cells sensitive to growth on medium containing 6-azauracil. RNA polymerase II purified from three different 6-azauracil-sensitive yeast strains displayed increased arrest at well characterized arrest sites in vitro. The extent of this defect did not correlate 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 eukaryotic RNA polymerases that is associated with nucleotide binding. The average elongation rate of this mutant enzyme is also slower than wild type. We suggest that the slowed elongation rate and an increase in dwell time of elongating pol II leads to rpb2 10's arrest-prone phenotype. This mutant enzyme can respond to SII for transcriptional read-through and carry out SII-activated nascent RNA cleavage.
Highlights
RNA polymerase II1 can encounter a variety of transcriptional blocks during the elongation phase of RNA synthesis
Arrest and Read-through of Mutant polymerase II (pol II)—Because 6AU sensitivity may reflect a defect in transcriptional elongation (Archambault et al, 1992; Exinger and Lacroute, 1992), we examined the ability of each mutant enzyme to overcome arrest in a region of a human histone gene (Reines et al, 1987)
In this study we have identified three mutant alleles of the second largest subunit of yeast pol II, rpb2– 4, rpb2–7, and rpb2–10 (Scafe et al, 1990a, 1990b), that confer sensitivity to growth of cells in the presence of 6AU and yield defective pol II enzymes
Summary
RNA polymerase II (pol II) can encounter a variety of transcriptional blocks during the elongation phase of RNA synthesis. The 6AU sensitivity caused by each of these mutant alleles can be complemented by overexpression of SII (Archambault et al, 1992), providing in vivo evidence for a role of SII in pol II transcription and suggesting that depression of NTP levels increases the reliance of the enzyme upon SII. These findings have suggested that 6AU sensitivity might serve as a bioassay for mutations in components of the elongation machinery, a direct demonstration that 6AU-sensitive RPB1 alleles generate an elongation-defective enzyme has not been reported
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