Mutations in the Second Largest Subunit of RNA Polymerase II Cause 6-Azauracil Sensitivity in Yeast and Increased Transcriptional Arrest in Vitro

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.RNA polymerase II (pol II) 1 can encounter a variety of transcriptional blocks during the elongation phase of RNA synthesis. These include DNA sequences (reviewed in , DNA bound proteins (Deuschle et al., 1990;Izban and Luse, 1991;Kuhn et al., 1990;Reines and Mote, 1993), DNA binding drugs (Mote et al., 1994), and covalent modifications due to DNA damage (Donahue et al., 1994). In vitro studies have identified mechanisms by which elongating pol II can overcome some of these obstacles. Intrinsic DNA sequences that cause pol II to become arrested have been most extensively investigated and are frequently employed as a model in studies of transcriptional arrest.Stably arrested pol II can be reactivated for RNA chain extension by elongation factor SII. SII binds the enzyme and activates a ribonuclease activity thought to reside within pol II (reviewed in ). This ribonuclease removes a small number of nucleotides from the 3′ end of the nascent RNA prior to its re-extension through the blockage Luse, 1993a, 1993b;Gu and Reines, 1995b) in a reiterative cleavage and resynthesis process, allowing a pol II molecule multiple attempts at chain extension through an obstacle (Gu et al

Histone Variant H2A.Z and RNA Polymerase II Transcription Elongation

“…Previous systematic pharmacogenetic screens had revealed that htz1⌬ single mutants have decreased resistance to 6-azauracil and mycophenolic acid (MPA) (17,82). By inhibiting inosine 5Ј-monophosphate (IMP) dehydrogenase, both of these drugs decrease the pool of ribonucleotides in vivo, which is not well tolerated in mutants with known defects in transcription elongation (21,35,61,64,76). Thus, the hypersensitivity of the single htz1⌬ mutant to these agents is potentially strong evidence for a role in elongation.…”

Interaction between Transcription Elongation Factors and mRNA 3′-End Formation at the Saccharomyces cerevisiae GAL10-GAL7 Locus

“…Frequently, elongation factor mutants are hypersensitive to 6-AU, presumably because they are less tolerant to 6-AU-induced pausing. An rpb2-7 mutant is strongly sensitive to 6-AU but shows phenotypes distinct from rpb1-221 and rpb2-10 (74,76,77). Fig.…”

The Paf1 complex is required for efficient transcription elongation by RNA polymerase I

“…These gaps likely reflect sites of paused complexes. Increased susceptibility to pausing/arresting has been shown to correlate with slower elongation rates (36).…”