Suppressor mutations in Escherichia coli RNA polymerase alter transcription initiation but do not affect translesion RNA synthesis in vitro

Abstract

Bacterial RNA polymerase (RNAP) coordinates transcription with DNA repair and replication. Many RNAP mutations have pleiotropic phenotypes with profound effects on transcription-coupled processes. One class of RNAP mutations (rpo∗) has been shown to suppress mutations in regulatory factors responsible for changes in gene expression during stationary phase or starvation, as well as in factors involved in the restoration of replication forks after DNA damage. These mutations were suggested to affect the ability of RNAP to transcribe damaged DNA and to decrease the stability of transcription complexes, thus facilitating their dislodging during DNA replication and repair, although this was not explicitly demonstrated. Here, we obtained nine mutations of this class located around the DNA/RNA binding cleft of Escherichia coli RNAP and analyzed their transcription properties in vitro. We found that these mutations decreased promoter complex stability to varying degrees, and all decreased the activity of rRNA promoters. However, they did not have strong effects on elongation complex stability. Some mutations were shown to stimulate transcriptional pauses or decrease intrinsic RNA cleavage by RNAP, but none altered the ability of RNAP to transcribe DNA templates containing damaged nucleotides. Thus, we conclude that the suppressor phenotypes of the mutations are unlikely to result from direct effects on DNA lesion recognition by RNAP but may be primarily explained by changes in transcription initiation. Further analysis of the effects of these mutations on the genomic distribution of RNAP and its interactions with regulatory factors will be essential for understanding their diverse phenotypes in vivo.