Transcriptional proofreading in Escherichia coli

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.