Temperature-Sensitive Mutants of RNase E in Salmonella enterica

Abstract

RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly663Cys, Ile2073Ser, Ile2073Asn, and Ala3273Pro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.RNase E (18) is a key component of the RNA degradosome (5, 20), a multienzyme complex that plays a central role in mRNA turnover and the processing of stable RNA in eubacteria (2,10,17,24,26). The degradosome is composed of a tetramer of RNase E molecules interacting via their N-terminal regions, with the C-terminal end of each RNase E molecule complexed with a monomer of RhlB, a dimer of enolase, and a trimer of PNPase (reviewed in reference 5). RNase E endonuclease activity can also be directed to specific mRNAs by the combined activity of Hfq protein and mRNA-specific small RNAs (13,25,31). The catalytic activity of RNase E is largely contained within the N-terminal half of the protein, the structure of which has been determined in Escherichia coli (4,10,15,22). The catalytic half of RNase E is divided into the small and big domains, with the latter including several folds related to RNase H, S1, and DNase I structures (4).In a previous study (11), mutations in RNase E were isolated in a selection for suppression of an extreme slow-growth phenotype caused by a mutant form of EF-Tu (tufA499, EF-Tu Gln125Arg) in Salmonella enterica serovar Typhimurium, and the question of how these mutations in RNase E suppress the tufA499 growth defect was raised. EF-Tu brings aminoacyltRNAs (aa-tRNAs), as part of the ternary complex EFTu ⅐ GTP ⅐ aa-tRN...

Turnover of mRNAs is one of the essential functions of RNase E

“…Here we addressed whether the degradation of one or more mRNAs might be an additional essential activity of RNase E. We previously isolated temperature‐sensitive Salmonella mutants with altered rne alleles that have a major effect on mRNA degradation at the non‐permissive temperature (Hammarlof et al ., ). The approach we took here was to identify external suppressors of these ts mutations in the expectation that their identity would reveal important insights into the essential functions of RNase E. Among 15 independent external suppressors that we isolated, the majority mapped within relBE whose only known activity is mRNA cleavage within the ribosomal A‐site.…”

Using the power of genetic suppressors to probe the essential functions of RNase E

“…We had previously identified four different rne mutations in Salmonella with a conditional lethal phenotype (temperature-sensitivity, ts) associated with a major reduction in mRNA turnover at the non-permissive temperature (Hammarlof et al 2011). Because these ts mutations were also associated with some (relatively minor) defects in rRNA and tRNA processing we could not draw any definitive conclusion regarding which of the RNase E activities were essential.…”

“…We isolated approximately 80 independent mutants capable of growth at the non-permissive temperature. Most of these carried second-site (intragenic) mutations in rne that compensated for the effect of the original ts mutation on structure-function relationships within RNase E (Hammarlof et al 2011). However, we also identified 15 independent mutants (selected to suppress rne-6 and rne-9, two of the four ts mutations) that retained the original ts allele in rne and carried in addition an external mutation that suppressed the conditional lethal phenotype (Hammarlof et al 2015).…”

“…RNase E is a bacterial single-strand-specific endonuclease (Cormack and Mackie 1992;Ehretsmann et al 1992) encoded by the essential gene, rne, in Escherichia coli (McDowall and Cohen 1996;Taraseviciene et al 1991) and Salmonella typhimurium (Hammarlof et al 2011). RNase E is the essential catalytic component of a multienzyme RNA degradasome complex (Carpousis 2007;Marcaida et al 2006) that is responsible for initiating the turnover of most mRNAs (Babitzke and Kushner 1991;Hammarlöf and Hughes 2008;Hammarlof et al 2011;Melefors and von Gabain 1991;Mudd et al 1990;Taraseviciene et al 1991), some sRNAs (Viegas et al 2007), and for processing stable RNAs such as 9S rRNA and most tRNAs, to their mature active forms (Apirion and Lassar 1978;Ghora and Apirion 1978;Hammarlof et al 2011;Li and Deutscher 2002;Lin-Chao et al 1999;Lundberg and Altman 1995;Ow and Kushner 2002;Viegas et al 2007).…”

A structural and biochemical comparison of Ribonuclease E homologues from pathogenic bacteria highlights species-specific properties

“…For example, the endoribonuclease RNase E is essential for cell viability in Escherichia coli 5–7 where roles in mRNA decay and the maturation of tRNA and rRNA have been well documented [reviewed in 1 and 8 ]. RNase E is also required for cell viability in Salmonella enterica 9 and has been implicated in the pathogenicity of both S. enterica and Yersinia pestis 10,11 . Furthermore, since homologues of RNase E are predicted to be present in many bacteria 12 , including pathogenic species, but are not found in animals or humans, RNase E is an ideal potential antibacterial target 2–4,13 .…”