title: Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.
abstract: Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity.
authors.affiliation: National Cancer Institute, National Institutes of Health, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: Yan Ning
authors.initials: YN
authors.lastname: Zhou
authors.forename: Lucyna
authors.initials: L
authors.lastname: Lubkowska
authors.forename: Monica
authors.initials: M
authors.lastname: Hui
authors.forename: Carolyn
authors.initials: C
authors.lastname: Court
authors.forename: Shuo
authors.initials: S
authors.lastname: Chen
authors.forename: Donald L
authors.initials: DL
authors.lastname: Court
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Strathern
authors.forename: Ding Jun
authors.initials: DJ
authors.lastname: Jin
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kashlev
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002875:Chromosomes / Q000648:ultrastructure; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007763:Lac Operon; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; D012333:RNA, Messenger; C540081:rpoB protein, E coli; D012321:DNA-Directed RNA Polymerases