title: Transcriptional proofreading in Escherichia coli.
abstract: A novel transcriptional proofreading mechanism associated with the beta-subunit of wild-type RNA polymerase from Escherichia coli is suggested from the following data. The purified holoenzyme contains an NTPase activity which specifically converts noncognate NTPs to their corresponding NDP in a template-dependent manner during in vitro transcription of synthetic single- and double-stranded templates. In contrast, purified enzyme from an rpoB mutant which shows increased transcriptional error lacked template-dependent NTP hydrolytic activity. The NTP hydrolytic activity of wild-type enzyme was critically dependent on the integrity of the initiation complex, and required continued transcriptional elongation. Transcription and translation of the lacZ gene proceeded 17% faster in the mutant than in its wild-type parent. These results are discussed in terms of a proofreading model in which the rate of transcription is limited by proofreading events that involve recognition and hydrolysis of noncognate NTPs before they can be misincorporated into RNA.
authors.affiliation: University of Washington, Department of Genetics, Seattle 98195.
authors.forename: R T
authors.initials: RT
authors.lastname: Libby
authors.forename: J L
authors.initials: JL
authors.lastname: Nelson
authors.forename: J M
authors.initials: JM
authors.lastname: Calvo
authors.forename: J A
authors.initials: JA
authors.lastname: Gallant
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D006868:Hydrolysis; D007700:Kinetics; D007763:Lac Operon; D009154:Mutation; D043583:Nucleoside-Triphosphatase; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012265:Ribonucleotides / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D012265:Ribonucleotides; D012321:DNA-Directed RNA Polymerases; D010744:Phosphoric Monoester Hydrolases; D043583:Nucleoside-Triphosphatase