Mutations of the beta subunit of RNA polymerase alter both transcription pausing and transcription termination in the trp operon leader region in vitro

Abstract

RNA polymerase was purified from rifampicin-resistant mutants of Escherichia coli which exhibit altered transcription termination at the trp operon attenuator in vivo. These mutant polymerases were used to investigate transcription pausing at the trp leader pause site and transcription termination at the trp attenuator. The mutant polymerases examined in vitro mimic their in vivo termination responses; i.e. RNA polymerase isolated from a mutant which displays high transcriptional read-through of the trp operon in vivo allows greater transcriptional read-through in vitro, while RNA polymerase prepared from a mutant which has reduced read-through in vivo exhibits greater termination of transcription in vitro. The observed differences are not due to the presence of--or response to--alternate secondary structures in the trp leader transcript since deletion of the DNA segment corresponding to some of these alternate structures does not affect termination efficiency. The mutant polymerases also have comparable effects on the kinetics of transcription pausing at the trp leader pause site; the termination-deficient polymerase exhibits diminished pausing while the termination-proficient polymerase displays enhanced pausing. We suggest that this correlation reflects polymerase recognition of similar features of RNA secondary structures in the pause and termination events. In addition, since single mutational changes in RNA polymerase affect two activities, pausing and termination, it is likely that a single site or region of the polymerase is involved in both events.