RESUMEN
CRISPR-associated Rossmann fold (CARF) domain signaling underpins modulation of CRISPR-Cas nucleases; however, the RtcR CARF domain controls expression of two conserved RNA repair enzymes, cyclase RtcA and ligase RtcB. Here, we demonstrate that RtcAB are required for RtcR-dependent transcription activation and directly bind to RtcR CARF. RtcAB catalytic activity is not required for complex formation with CARF, but is essential yet not sufficient for RtcRAB-dependent transcription activation, implying the need for an additional RNA repair-dependent activating signal. This signal differs from oligoadenylates, a known ligand of CARF domains, and instead appears to originate from the translation apparatus: RtcB repairs a tmRNA that rescues stalled ribosomes and increases translation elongation speed. Taken together, our data provide evidence for an expanded range for CARF domain signaling, including the first evidence of its control via in trans protein-protein interactions, and a feed-forward mechanism to regulate RNA repair required for a functioning translation apparatus.
RESUMEN
RNA ligases function pervasively across the three kingdoms of life for RNA repair, splicing and can be stress induced. The RtcB protein (also HSPC117, C22orf28, FAAP and D10Wsu52e) is one such conserved ligase, involved in tRNA and mRNA splicing. However, its physiological role is poorly described, especially in bacteria. We now show in Escherichia coli bacteria that the RtcR activated rtcAB genes function for ribosome homeostasis involving rRNA stability. Expression of rtcAB is activated by agents and genetic lesions which impair the translation apparatus or may cause oxidative damage in the cell. Rtc helps the cell to survive challenges to the translation apparatus, including ribosome targeting antibiotics. Further, loss of Rtc causes profound changes in chemotaxis and motility. Together, our data suggest that the Rtc system is part of a previously unrecognized adaptive response linking ribosome homeostasis with basic cell physiology and behaviour.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fenotipo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Quimiotaxis , Escherichia coli/inmunología , Sitios Genéticos , Homeostasis , Operón , Biosíntesis de Proteínas , ARN Ribosómico , Ribosomas/metabolismoRESUMEN
This article describes reproducibility of a single-step automated ß-galactosidase, and the equivalence of its data to the traditional assay ("Experiments in Molecular Genetics" [1]). This was done via a pairwise comparison of both methods using strains with Miller Unit [MU] values ranging from 0 to over 2000. The data presented in this article is associated with the research article entitled "A single-step method for mid to high throughput ß-galactosidase assays in Escherichia coli using a microplate reader" [2].
RESUMEN
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%.
Asunto(s)
Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Escherichia coli/enzimología , beta-Galactosidasa/metabolismo , Células Cultivadas , Escherichia coli/citología , beta-Galactosidasa/análisisRESUMEN
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.