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: Gly66→Cys, Ile207→Ser, Ile207→Asn, and Ala327→Pro. 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.

Fig. 1.

(Left) The catalytic part of a monomer of RNase E. There are five domains or subdomains in the catalytic part, and they are marked with unique colors. The RNase H subdomain is composed of two chain segments and has two colors. This drawing is based on PDB entry 2bx2 (4). (Right) Structure of an RNase E tetramer using the same color scheme as the left panel. Three of the subunits are shown in pale colors. A double-stranded RNA oligonucleotide (red) is included in this drawing, based on PDB entry 2c0b.

Fig. 2.

Mutations in the RNA-binding region of RNase E that suppress the slow-growth phenotype caused by the EF-Tu mutation tufA499. The coloring is the same as in Fig. 1, but the orientation is different. The side chains shown are those of the nonmutated E. coli structure in PDB entry 2c4r (4). The active-site residues Asp303 and Asp346, shown with bound Mg²⁺ ion (yellow) and water molecules (red spheres) binding to the metal ion, are also included. The DNase I subdomain from another subunit in the tetramer is also included.

Fig. 3.

Some of the mutations in the small domain of RNase E. (Left) The contact area between the RNase H subdomain of one subunit and the small domain, including the linker, is shown. (Right) The contact of two small domains. This interaction stabilizes the tetramer formation of two dimers, and several polar residues in the contact area are mutated in the suppressors. Drawings are based on PDB entry 2bx2 (4).

Fig. 4.

Suppressor mutations for the rne-6 (Ile207Ser) and rne-9 (Ile207Asn) temperature-sensitive mutations. The coloring is the same as in Fig. 1. The suppressor mutations are found both in the 5′ sensor subdomain and in the RNase H and DNase I subdomains. The drawing is based on PDB entry 2bx2 (4).

Fig. 5.

Suppressor mutations for the rne-10 (left) and rne-11 (right) temperature-sensitive mutations. The coloring is the same as in Fig. 1. The suppressor mutations for rne-10 are close to the original mutations or in the 5′ sensor subdomain. The suppressors for rne-11 are all within the DNase 1 subdomain. The drawing is based on PDB entry 2bx2 (4).

Fig. 6.

Relative growth rates of the wild type (WT), four different rne-TS mutants, and rne-TS⁺ suppressor double mutants in LB broth as a function of growth temperature. The growth rate of the wild type at each temperature is set to 1.

Fig. 7.

Steady-state levels of tufA, fusA, and rne mRNA. Strains TH7589, TH7599, TH7590, TH7602, TH7591, and TH7612 were grown at 30°C until the OD₆₀₀ was 0.2 to 0.4 and then shifted to 43°C for 30 min. Total RNA was isolated from cultures grown at 30°C and from cultures shifted to 43°C. The steady-state levels of the tufA, fusA, and rne mRNAs were quantified by RT-PCR. WT, wild type.

Fig. 8.

Processing of 9S to 5S rRNA in TS and TS suppressor strains. Strains TH7589, TH7599, TH7590, TH7602, TH7591, and TH7612 were grown at 30°C until the OD₆₀₀ was 0.2 to 0.4 and then shifted to 43°C for 30 min. Total RNA was isolated from cultures grown at 30°C and 43°C, separated on a 12% polyacrylamide gel, and probed for 5S. (A) Representative gel showing incomplete processing in strains with mutations in rne after growth at 43°C. Bands corresponding to 9S and 5S are indicated. (B), Percent incompletely processed 9S rRNA for the wild type (wt) and different rne mutants.