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CRISPR-associated Rossmann fold (CARF) domain signalling underpins the modulation of CRISPR-Cas systems. Unlike the majority of known CARF domains associated with nuclease activity in CRISPR-Cas systems, the CARF domain of the transcriptional regulator RtcR modulates the opposite function by activating an RNA end sealing system. The Rtc RNA repair system in Escherichia coli consists ofthe universally conserved RNA cyclase RtcA and RNA ligase RtcB, and is known to be induced by antibiotics and oxidative stress. We aim to investigate the CARF domain mediated transcriptional regulation of the Rtc system in vivo and in vitro. A reporter based assay confirmed that the RtcR CARF domain has a negative regulatory effect on RtcR activity and subsequent induction of the rtcBA operon. Both predictive modelling and site-directed mutagenesis suggest the Rtc induction is not due to oxidation of cysteines present in the RtcR regulatory CARF domain. Interestingly, the enzymes RtcA and RtcB, products of the actions of RtcR as a transcription regulator, are required for RtcR to stimulate expression of the rtcBA operon and directly bind to the RtcR CARF domain as shown in vivo by two-hybrid and in vitro by gel filtration. Given that CARF domains are known to be activated by RNA binding, a range of RNA molecules linked to the Rtc system or CARF domains in general are currently being investigated as potential inducers in an in vitro transcription system. Together, our data indicate an expanded range for CARF domain signalling, including via protein-protein interactions.

Enhancer-dependent transcription involving the promoter specificity factor σ(54) is widely distributed amongst bacteria and commonly associated with cell envelope function. For transcription initiation, σ(54)-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ(54) promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ(54) were evident, indicating loss of rpoN (encoding σ(54)) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ(54)-dependence in vivo not readily correlated with conventional σ(54) binding-sites.

Historically, the lacZ gene is one of the most universally used reporters of gene expression in molecular biology. Its activity can be quantified using an artificial substrate, o-nitrophenyl-ß-d-galactopyranoside (ONPG). However, the traditional method for measuring LacZ activity (first described by J. H. Miller in 1972) can be challenging for a large number of samples, is prone to variability, and involves hazardous compounds for lysis (e.g., chloroform, toluene).Here we describe a single-step assay using a 96-well microplate reader with a proven alternative cell permeabilization method. This modified protocol reduces handling time by 90%.