Autoregulation allows Escherichia coli RNase E to adjust continuously its synthesis to that of its substrates

The Escherichia coli endonuclease RNase E plays a key role in rRNA maturation and mRNA decay. In particular, it controls the decay of its own mRNA by cleaving it within the 5'-untranslated region (UTR), thereby autoregulating its synthesis. Here, we report that, when the synthesis of an RNase E substrate is artificially induced to high levels in vivo, both the rne mRNA concentration and RNase E synthesis increase abruptly and then decrease to a steady-state level that remains higher than in the absence of induction. Using rne-lacZ fusions that retain or lack the rne 5'UTR, we show that these variations reflect a transient mRNA stabilization mediated by the rne 5'UTR. Finally, by putting RNase E synthesis under the control of an IPTG-controlled promoter, we show that a similar, rne 5'UTR-mediated mRNA stabilization can result from a shortage of RNase E. We conclude that the burst in substrate synthesis has titrated RNase E, stabilizing the rne mRNA by protecting its 5'UTR. However, this stabilization is self-correcting, because it allows the RNase E pool to expand until its mRNA is destabilized again. Thus, autoregulation allows RNase E to adjust its synthesis to that of its substrates, a behaviour that may be common among autoregulated proteins. Incidentally, this adjustment cannot occur when translation is blocked, and we argue that the global mRNA stabilization observed under these conditions originates in part from this defect.     

RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli.

We have isolated suppressor mutants that suppress temperature-sensitive colony formation and anucleate cell production of a mukB mutation. A linkage group (smbB) of the suppressor mutations is located in the rne/ams/hmp gene encoding the processing endoribonuclease RNase E. All of the rne (smbB) mutants code for truncated RNase E polypeptides lacking a carboxyl-terminal half. The amount of MukB protein was higher in these rne mutants than that in the rne+ strain. These rne mutants grew nearly normally in the mukB+ genetic background. The copy number of plasmid pBR322 in these rne mutants was lower than that in the rne+ isogenic strain. The results suggest that these rne mutations increase the half-lives of mukB mRNA and RNAI of pBR322, the antisense RNA regulating ColE1-type plasmid replication. We have demonstrated that the wild-type RNase E protein bound to polynucleotide phosphorylase (PNPase) but a truncated RNase E polypeptide lacking the C-terminal half did not. We conclude that the C-terminal half of RNase E is not essential for viability but plays an important role for binding with PNPase. RNase E and PNPase of the multiprotein complex presumably cooperate for effective processing and turnover of specific substrates, such as mRNAs and other RNAs in vivo.     

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.     

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.     

Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA

Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo. Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180kDa. However, proteolytic fragments as small as 70kDa, which can arise during purification, also exhibit RNase E activity, in vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites. RNase E cleaves rne mRNA and autoregulates the expression of the rne gene. In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cieaved by RNase K. Our data are consistent with RNase K being a proteolytic fragment of RNase E.     

Temperature-sensitive mutants of RNase E in Salmonella enterica

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.     

Processing of bacteriophage T4 tRNAs: a precursor of species 1 RNA

A precursor molecule of species 1 RNA, p2Sp1, that accumulates when an rne (RNase E-) mutant is infected with a T4 deletion mutant (delta 27) is also found after infection of an rne host mutant by different deletion mutants or wild type bacteriophage T4. Low levels of this molecule were also found in a wild-type host infected with a wild-type T4. This precursor molecule accumulates at higher concentrations at 43 degrees C as compared to 30 degrees C or 37 degrees C. Structural analysis of the precursor molecules from the different sources has shown a complete identity of p2Sp1 RNA isolated from the different sources. Therefore, we suggest that this precursor is a normal intermediate in processing of T4 tRNAs, and that it is unrelated to a particular T4 deletion strain. Since RNase E does not process this precursor, its accumulation in an rne mutant reflects an interaction between RNase E and the enzyme that processes this intermediate.