RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue,RNase E

RNase G (rng) is an E. coli endoribonuclease that is homologous to the catalytic domain of RNase E (rne), an essential protein that is a major participant in tRNA maturation, mRNA decay, rRNA processing and M1 RNA processing. We demonstrate here that whereas RNase G inefficiently participates in the degradation of mRNAs and the processing of 9S rRNA, it is not involved in either tRNA or M1 RNA processing. This conclusion is supported by the fact that inactivation of RNase G alone does not affect 9S rRNA processing and only leads to minor changes in mRNA half-lives. However, in rng rne double mutants mRNA decay and 9S rRNA processing are more defective than in either single mutant. Conversely, increasing RNase G levels in an rne-1 rng::cat double mutant, proportionally increased the extent of 9S rRNA processing and decreased the half-lives of specific mRNAs. In contrast, variations in the amount of RNase G did not alter tRNA processing under any circumstances. Thus, the failure of RNase G to complement rne mutations, even when overproduced at high levels, apparently results from its inability to substitute for RNase E in the maturation of tRNAs.     

A novel RNase G mutant that is defective in degradation of adhE mRNA but proficient in the processing of 16S rRNA precursor.

Escherichia coli RNase G, encoded by the rng gene, is involved in both the processing of 16S rRNA precursor and the degradation of adhE mRNA. Consequently, defects in RNase G result in elevation of AdhE levels. Furthermore, the adhR430 mutant strain, DC430, is reported to overproduce the AdhE protein in a manner dependent on the adhC81 mutation. We found that overproduction of AdhE by DC430 was reversed to wild-type levels by introduction of a plasmid carrying the wild-type allele of rng. Mapping by P1-phage-mediated transduction also indicated that a mutation involved in AdhE overproduction was located around the rng region in DC430. DNA sequencing of the rng region revealed that DC430 indeed had a mutation in the rng gene: a G1022 to A transition that caused substitution of Gly341 with Ser and which was named rng430. This lies in the highly conserved region of the RNase E/RNase G family, called high similarity region 2 (HSR2). However, very interestingly, rng430 mutant strains did not accumulate the 16.3S precursor of 16S rRNA unlike rng::cat mutants. We also found that the Rng1 mutant protein, which is truncated in its C-terminal domain encompassing HSR2, exhibited a residual processing activity against the 16S rRNA precursor, when overproduced. These results indicate that the HSR2 of RNase G plays an important role in substrate recognition and/or ribonucleolytic action.     

Second-site suppression of RNase E essentiality by mutation of the deaD RNA helicase in Escherichia coli

Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.     

A Streptomyces coelicolor functional orthologue of Escherichia coli RNase E shows shuffling of catalytic and PNPase-binding domains

Previous work has detected an RNase E-like endoribonucleolytic activity in cell extracts obtained from Streptomyces. Here, we identify a Streptomyces coelicolor gene, rns, encoding a 140 kDa protein (RNase ES) that shows endoribonucleolytic cleavage specificity characteristic of RNase E, confers viability on and allows propagation of Escherichia coli cells lacking RNase E and accomplishes RNase E-like regulation of plasmid copy number in E. coli. However, notwithstanding its complementation of rne-deleted E. coli, RNase ES did not accurately process 9S rRNA from E. coli. Additionally, whereas RNase E is normally required for E. coli survival, rns is not an essential gene in S. coelicolor. Deletion analysis mapped the catalytic domain of RNase ES near its centre and showed that regions located near the RNase ES termini interact with an S. coelicolor homologue of polynucleotide phosphorylase (PNPase) - a major component of E. coli RNase E-based degradosomes. The interacting arginine- and proline-rich segments resemble the C-terminally located degradosome scaffold region of E. coli RNase E. Our results indicate that RNase ES is a structurally shuffled RNase E homologue showing evolutionary conservation of functional RNase E-like enzymatic activity, and suggest the existence of degradosome-like complexes in Gram-positive bacteria.     

The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.     

A developmentally regulated Streptomyces endoribonuclease resembles ribonuclease E of Escherichia coli

We report that the Streptomyces species S. lividans and S. coelicolor , morphologically complex Gram-positive soil bacteria, contain a developmentally regulated endoribonuclease activity (here named RNase ES) that functionally and immunologically resembles Escherichia coli RNase E. In Streptomyces cells, RNA I — the antisense repressor of replication of ColE1-type plasmids — is cleaved at sites attacked by RNase E. A Mg²⁺-dependent endonuclease that produces RNase E-like cleavages in RNA I and 9S ribosomal RNA was identified in S. lividans cell extracts. A Streptomyces peptide migrating at 70 kDa in SDS/polyacrylamide gels binds to RNase E substrates and reacts with three separate anti-RNase E monoclonal antibodies; the endonucleolytic cleavage activity co-purified with the immunoreactive 70 kDa peptide. We show that RNase ES activity is regulated during the Streptomyces life cycle: activity increased as cells progressed from exponential growth to stationary phase in liquid culture, or from mycelial growth to sporulation on solid media. While mutations that interfere with S. coelicolor development late in its life cycle did not prevent this developmentally associated increase in RNase ES activity, the increase was blocked by a mutation ( bldA ) that interferes early with both morphological and physiological differentiation.     

A Streptomyces coelicolor Antibiotic Regulatory Gene, absB, Encodes an RNase III Homolog

Streptomyces coelicolor produces four genetically and structurally distinct antibiotics in a growth-phase-dependent manner. S. coelicolor mutants globally deficient in antibiotic production (Abs(-) phenotype) have previously been isolated, and some of these were found to define the absB locus. In this study, we isolated absB-complementing DNA and show that it encodes the S. coelicolor homolog of RNase III (rnc). Several lines of evidence indicate that the absB mutant global defect in antibiotic synthesis is due to a deficiency in RNase III. In marker exchange experiments, the S. coelicolor rnc gene rescued absB mutants, restoring antibiotic production. Sequencing the DNA of absB mutants confirmed that the absB mutations lay in the rnc open reading frame. Constructed disruptions of rnc in both S. coelicolor 1501 and Streptomyces lividans 1326 caused an Abs(-) phenotype. An absB mutation caused accumulation of 30S rRNA precursors, as had previously been reported for E. coli rnc mutants. The absB gene is widely conserved in streptomycetes. We speculate on why an RNase III deficiency could globally affect the synthesis of antibiotics.     

Functional implications of the conserved action of regulators of ribonuclease activity

RNase E (Rne) plays a major role in the decay and processing of numerous RNAs in E. coli, and protein inhibitors of RNase E, RraA and RraB, have recently been discovered. Here, we report that coexpression of RraA or RraB reduces the ribonucleolytic activity in rne-deleted E. coli cells overproducing RNase ES, a Streptomyces coelicolor functional ortholog of RNase E, and consequently rescues these cells from growth arrest. These findings suggest that the regulators of ribonuclease activity have a conserved intrinsic property that effectively acts on an RNase E-like enzyme found in a distantly related bacterial species.     

RNA processing: new mutants that affect endonucleolytic processing of RNA

A strain of Escherichia coli carrying the rne-3071 mutation that affects the RNA processing enzyme ribonuclease E, was mutagenized, and double mutants deficient in RNA processing were isolated. The isolation was based on the appearance of a particular RNA precursor molecule upon infection of an rne mutant with a specific bacteriophage T4 deletion strain. From one of the double mutants the rne mutation was removed, and the new single mutant, designated rng, was examined. In this mutant the maturation of host RNA as well as of bacteriophage T4 RNA is affected. The effect of the rng mutation on RNA synthesis is unique and can be distinguished from the effects of the other established mutations in RNA processing. The effects of the rng mutation can be recognized in vivo and in vitro.     

RNase G-dependent degradation of the eno mRNA encoding a glycolysis enzyme enolase in Escherichia coli

Escherichia coli RNase G, encoded by the rng gene, is involved in the processing of 16S rRNA and degradation of the adhE mRNA encoding a fermentative alcohol dehydrogenase. In a search for the intracellular target RNAs of RNase G other than the 16S rRNA precursor and adhE mRNA, total cellular proteins from rng+ and rng::cat cells were compared by two-dimensional gel electrophoresis. The amount of enolase encoded by the eno gene reproducibly increased two- to three-fold in the rng::cat mutant strain compared with the rng+ parent strain. Rifampicin chase experiments showed that the half-life of the eno mRNA was some 3 times longer in the rng::cat mutant than in the wild type. These results indicate that the eno mRNA was a substrate of RNase G in vivo, in addition to 16S rRNA precursor and adhE mRNA.     

Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor.

A homolog of the mmsA gene of Pseudomonas aeruginosa, which encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is involved in valine catabolism in pseudomonads and mammals, was cloned and sequenced from Streptomyces coelicolor. Of the two open reading frames (ORFs) found, which are convergently transcribed and separated by a 62-nucleotide noncoding region, the deduced amino acid sequence of the msdA ORF (homologous to mmsA) is similar to a variety of prokaryotic and eukaryotic aldehyde dehydrogenases that utilize NAD+, particularly to the MmsA protein from P. aeruginosa. No significant similarity was found between the deduced product of ORF1 and known proteins in the databases. An S. coelicolor msdA mutant, constructed by insertion of a hygromycin resistance gene (hyg) into the msdA coding region, lost the MSDH activity and the ability to grow in a minimal medium with valine or isobutyrate as the sole carbon source but grew on propionate. The msdA::hyg mutation was complemented by introduction of the msdA gene on a plasmid. When the S. coelicolor msdA gene was overexpressed in Escherichia coli under the control of the T7 promoter, a protein of 51-kDa, corresponding to the approximate mass of the predicted S. coelicolor msdA product (52.6 kDa), and specific MSDH activity were detected. These results strongly suggest that msdA indeed encodes the MSDH that is involved in valine catabolism in S. coelicolor.     

NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD(+)-dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD(+) as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperature-sensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD(+)-dependent DNA ligases.