The long-standing assumption that messenger RNA (mRNA) degradation in Escherichia coli begins with endonucleolytic cleavage has been challenged by the recent discovery that RNA decay can be triggered by a prior non-nucleolytic event that marks transcripts for rapid turnover: the rate-determining conversion of the 5' terminus from a triphosphate to a monophosphate. This modification creates better substrates for the endonuclease RNase E, whose cleavage activity at internal sites is greatly enhanced when the RNA 5' end is monophosphorylated. Moreover, it suggests an explanation for the influence of 5' termini on the endonucleolytic cleavage of primary transcripts, which are triphosphorylated. However, no enzyme capable of removing pyrophosphate from RNA 5' ends has been identified in any bacterial species. Here we show that the E. coli protein RppH (formerly NudH/YgdP) is the RNA pyrophosphohydrolase that initiates mRNA decay by this 5'-end-dependent pathway. In vitro, RppH efficiently removes pyrophosphate from the 5' end of triphosphorylated RNA, irrespective of the identity of the 5'-terminal nucleotide. In vivo, it accelerates the degradation of hundreds of E. coli transcripts by converting their triphosphorylated 5' ends to a more labile monophosphorylated state that can stimulate subsequent ribonuclease cleavage. That the action of the pyrophosphohydrolase is impeded when the 5' end is structurally sequestered by a stem-loop helps to explain the stabilizing influence of 5'-terminal base pairing on mRNA lifetimes. Together, these findings suggest a possible basis for the effect of RppH and its orthologues on the invasiveness of bacterial pathogens. Interestingly, this master regulator of 5'-end-dependent mRNA degradation in E. coli not only catalyses a process functionally reminiscent of eukaryotic mRNA decapping but also bears an evolutionary relationship to the eukaryotic decapping enzyme Dcp2.

The lifetimes of bacterial mRNAs are strongly affected by their association with ribosomes. Events occurring at any stage during translation, including ribosome binding, polypeptide elongation, or translation termination, can influence the susceptibility of mRNA to ribonuclease attack. Ribosomes usually act as protective barriers that impede mRNA cleavage, but in some instances they can instead trigger the decay of the mRNA to which they are bound or send a signal that leads to widespread mRNA destabilization within a cell. The influence of translation on mRNA decay provides a quality-control mechanism for minimizing the use of poorly or improperly translated mRNAs as templates for the production of abnormal proteins that might be toxic to bacteria.

The common belief that endonucleolytic cleavage is the initial, rate-determining step of mRNA decay in Escherichia coli fails to explain the influence of 5' termini on the half-lives of primary transcripts. We have re-examined the initial events of RNA degradation in that organism by devising an assay to probe the 5' phosphorylation state of RNA and by employing a self-cleaving hammerhead ribozyme to investigate the degradative consequences of an unphosphorylated 5' end. These studies have identified a previously unrecognized prior step in decay that triggers subsequent internal cleavage by the endonuclease RNase E and thereby governs RNA longevity: the rate-determining conversion of a triphosphorylated to a monophosphorylated 5' terminus. Our findings redefine the role of RNase E in RNA degradation and explain how unpaired 5'-terminal nucleotides can facilitate access to internal cleavage sites within primary transcripts. Moreover, these results reveal a striking parallel between the mechanisms of mRNA decay in prokaryotic and eukaryotic organisms.

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SummaryThe endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10-20% of normal and that the feedback mechanism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the increase in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein. IntroductionMessenger RNA degradation is a key mechanism for controlling gene expression in all organisms. In Escherichia coli, the ribonuclease that appears to be most important in governing this process is the endonuclease RNase E, as thermal inactivation of this enzyme impedes the decay of bulk mRNA and of most individual transcripts (Apirion, 1978;Ono and Kuwano, 1979;Mudd et al., 1990a;Babitzke and Kushner, 1991;Melefors and von Gabain, 1991;Taraseviciene et al., 1991 temperature-sensitive rne alleles, ams-1 and rne-3071 (Apirion, 1978;Ono and Kuwano, 1979). These alleles encode mutant forms of RNase E that each bear a single amino acid substitution (McDowall et al., 1993). Even though these rne mutants were first isolated more than 20 years ago, the catalytic defects caused by the mutations are not understood. Furthermore, the temperaturesensitive nature of the mutants requires that they be studied immediately after a temperature upshift (i.e. in heat-shocked cells experiencing growth arrest) or at semi-permissive temperatures where the relative specific activity of these RNase E mutants is unknown. These uncertainties have limited the utility of the ams-1 and rne-3071 alleles for examining the effects of a dearth of RNase E activity in E. coli.To overcome these impediments, we have constructed an E. coli strain in which the cellular concentration of wildtype RNase E can be manipulated at will. This strain has enabled us to investigate the consequences of RNase E scarcity for RNA degradation, feedback regulation and E. coli cell growth and to test whether there are extragenic mutations that can restore growth to cells lacking RNase E. Results Construction and characterization of an E. coli strain bearing an inducible rne geneTo make it possible to examine the effect of reducing the cellular concentration of wild-type RNase E, we constructed an E. coli strain in which expression of ...