Regulation and biological function of the RNA ligase RtcB in Escherichia coli

Abstract

All organisms contain RNA ligases for the modification or repair of RNA molecules. The RtcB family have a novel 3ʹ-5ʹ RNA ligase activity and are conserved in all three domains of life. Metazoan homologues of RtcB have essential roles in tRNA maturation and the unfolded protein response. In E. coli, expression of RtcB from the σ54-dependent rtcBA operon is regulated by transcriptional activator RtcR. Neither the signals that induce rtcB expression nor the natural substrates of RtcB in E. coli are known. The objective of this project is to understand the regulation of rtcB expression by RtcR and the biological function of RtcB in E. coli. In this study, we confirm the roles of RtcR and σ54 in rtcB transcription in E. coli. Using a series of truncated rtcBA promoter fusions with the reporter gene lacZ, we have identified an inverted repeat that we hypothesise is the RtcR binding site in the promoter of rtcB. Mutation of the inverted repeat supports its involvement in RtcR binding. Using a similar approach, we have identified a possible σ70 promoter responsible for transcription of rtcR and provide evidence of negative autoregulation of rtcR. Moreover, we also demonstrate for the first time the requirement of IHF for RtcR-dependent transcription of rtcB and confirm the putative binding site of IHF in the rtcBA promoter in vivo and in vitro. Using a combination of genetic screening with a chromosomal rtcBA-lacZ fusion and quantitative PCR analysis, we have uncovered several conditions where transcription of rtcB is activated in E. coli. Expression of Rof, an inhibitor of Rho transcription termination, induced rtcB transcription in an RtcR-dependent manner, indicating a possible link between RtcB function and disruption of transcription termination. Furthermore, treatment of cells with chloramphenicol and induction of endoribonuclease MazF increased rtcB transcription, suggesting that RtcB may respond to the translational status of the cell. We have used these conditions to perform CRAC (crosslinking and analysis of cDNA) of RtcB, in order to identify substrate RNAs that interact with RtcB in vivo. Many interactions between RtcB and RNAs were identified including rRNAs, tRNAs, sRNAs and mRNAs and several candidates were further analysed by qPCR. Taken together, the data suggests that rtcB has features typical of a σ54-dependent promoter and that RtcB expression can be activated in a variety of stress conditions, thereby allowing interaction with a range of cellular RNAs in E. coli. Nevertheless, the specific signal(s) detected by RtcR and the biological function of RtcB in E. coli remain elusive.