rne

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..with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants...

..The rne gene was replaced with alleles encoding deletions in the non-catalytic part of RNase E...

The rne gene of Escherichia coli encodes a 118 kDa protein that has ribonuclease E (RNase E) activity and binds RNA...

..Thus, this large class of regulatory RNAs share an unexpected intrinsic mechanism for shutting off their action...

..coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels...

..Hfq accelerates the rate of duplex formation between SgrS and the target mRNA. Membrane localization of target mRNA contributes to efficient SgrS action by competing with ribosome loading...

..These results suggest that RhlB acts by unwinding RNA structures that impede the processive activity of PNPase. RhlB is thus an important enzyme in mRNA turnover...

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..We discuss the implications for the regulation of RNA degradosome function in vivo...

..coli, the phenomenon of 'all or none' mRNA decay, and the stabilization provided by 5' stem-loop structures...

..We conclude that 5' extremities modulate the poly(A)-dependent degradation of mRNA fragments and that the 5' cleavage by RNase E at M3 activates the chemical degradation of the rpsO mRNA...

..RhlB in vitro can facilitate the degradation of structured RNA by PNPase. Since the discovery of the RNA degradosome in E. coli, related complexes have been described in other organisms...

..By using several new rne alleles, we have confirmed these observations and have also ruled out that M1 processing by RNase E is required for ..

..One or both interfaces organize the active site, which is distinct from the primary site of RNA binding...

..Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E. coli, also suggest that RNA processing and decay may occur at specific sites within cells...

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..These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5' end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G...

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..coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3' end of ssrA RNA...

..Autoregulation is mediated in cis by the 361-nucleotide 5' untranslated region (UTR) of rne (RNase E) mRNA...

..The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5'-UTR...

..Conversely, toxicity is significantly reduced in the presence of excess RNase II. Overproduction of RNase E leads to increased polyadenylation and no reduction in toxicity...

..However, transiently increasing intracellular poly(A) levels has also been shown to stabilize the pnp and rne transcripts, leading to increased polynucleotide phosphorylase (PNPase) and RNase E levels respectively...

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...

..Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs...

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..coli...

..These results demonstrated that other ribonucleases can act as an additional level of regulation in the control of the expression of RNase R...

..One possible physiological role of mRNA degradation mediated by sRNP is to rid the cell of translationally inactive mRNAs, making gene silencing irreversible...

..The formation of ribonucleoprotein complexes containing RNases could be a general way by which small RNAs destabilize target mRNAs in both prokaryotes and eukaryotes...

..These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels...

..Selective conservation of the AUE is usually not observed in bacteria without RNase E. These results demonstrate a novel example of co-evolution of RNA sequences with processing activities...

..The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed...

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..In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell...

..We also discuss the role of poly(A)-dependent decay in mRNA metabolism...

..It is commonly believed that the RNA chaperone Hfq facilitates or promotes the interaction between sRNAs and their mRNA targets. This study reveals another role for Hfq, that is, protection of sRNAs from endonucleolytic attack...

..Among the molecular mechanisms that could account for these properties are those in which 5'-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit...

..Moreover, the simplicity and efficiency of the proposed assay suggest that it can be a valuable tool not only for the characterization of RNase E homologs but also for the analysis of other site-specific nucleases...

RNase E autoregulates its production in Escherichia coli by governing the decay rate of rne (RNase E) mRNA. It does so by a mechanism that is dependent in part on hp2, a cis-acting stem-loop within the rne 5' untranslated region...

..The implications of these findings for the cellular organization of the RNA-processing enzymes in the cell are discussed...

..Introduction of a cafA::cat mutation enhanced the temperature sensitivity of the ams1 mutant. These results suggest that there is a functional homology between these two proteins...

A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature...

..a series of multiple mutants containing the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and ams-1 (altered message stability) alleles, it was possible to study general mRNA turnover as well as the degradation of ..

..Although the effect of the rne temperature-sensitive mutation could be indirect, the simplest interpretation of our results is that RNase E acts ..

..The enzyme(s) involved in this degradation pathway is not known. We detected an unstable elongated rpsO mRNA presumably resulting from the addition of nucleotides at the 3' end of the transcript...

..Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure...

..The other two cleavages are dependent on the wild-type allele of the rne gene, which encodes the endonucleolytic enzyme RNase E...

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 180 kDa...

..In contrast polyadenylylation does not significantly modify the stability of rpsO mRNA undergoing RNase E-mediated degradation...

..The low beta-galactosidase yield from T7 transcripts reflects their low stability: the ams-1/rne-50 mutation, which inactivates RNase E, nearly equalizes the beta-galactosidase yields from T7 and E...

..We also show that Sok RNA is polyadenylated by poly(A) polymerase I (PAP I), and that the poly(A)-tailing is prerequisite for the rapid 3'-exonucleolytic degradation by PNPase...

..The half-lives of individual mRNAs were increased in both a delta pcnB single mutant and a delta pcnB pnp-7 rnb-500 rne-1 multiple mutant...

..We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent...

..Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites...

We have used an antibody to a previously identified 180 kDa (Hmp1) protein in Escherichia coli to clone the corresponding gene, which encodes a polypeptide of 114 kDa that has a mobility equivalent to 180 kDa in SDS/PAGE...

..Taken together, our results suggest that in wild-type E. coli the degradation of the RNase III processed mRNA is mediated by RNase E...

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..of precursor 5S ribosomal ribonucleic acid (RNA) from the nascent ribosomal RNA transcript, is thermolabile in rne-3071 mutants...

..These findings suggest that inability of cells over-producing RNase E to normally grow results from increased cellular ribonucleolytic activity and RraA is able to effectively modulate the catalytic activity of RNase E in vivo...

The activity, ribonuclease E, isolated from an rne mutant was irreversibly inactivated at lower temperatures than ribonuclease E isolated from a wild-type strain...

..We have sequenced a 3.2 kb EcoRI-BamHI fragment encoding the rne gene, and identified its reading frame...

The endoribonuclease RNase E, encoded by the essential gene rne, plays a major role in cellular RNA metabolism, i.e...

..We show that a representative dominant-negative mutant can form mixed multimers with RNase E and propose a model to explain how these mutants can block wild-type RNase E function in vivo...

..a twofold increase in beta-galactosidase activity from a chromosomal fusion of the 5' untranslated region of the rne gene to lacZ, suggesting that a reduction in cellular concentration of UDP-GlcNAc and the resulting increased ..

..We searched for proteins that showed changes in expression in both hfq::cat and rne-1 mutant cells as compared with the wild type, and found that a protein band of 49-kDa decreased in these mutant ..

..The endonuclease associated with this "killing" activity, which has a eukaryotic counterpart ("no-go decay"), is not characterized; it may be borne by the distressed ribosome itself...

..Most of the approaches that we describe can be applied to the study of homologs of E. coli RNase E, which have been found in approximately half of the eubacteria that have been sequenced...

..This chapter describes purification of RNase E (the Rne protein), reconstitution of a minimal degradosome that recapitulates the activity of authentic degradosomes, and ..

..Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments...

..Other ribonucleases, such as RNase III and MazF, do not play a role in pst mRNA processing. RNase E is thus at least partially responsible for processing the pst primary transcript...

..These findings shed light on important but largely unexplored aspects of cellular structure and function, including the organization of the RNA processing machinery of the cell and of bacterial cytoskeletal elements in general...

..Here we characterize a novel mutant in the rne promoter region, which regulates RNase E expression...

..coli cell cycle. We shall also briefly indicate recent data from other laboratories consistent with our general hypothesis...

..E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli...

..Our data also demonstrate that the Rne-1 protein retains significant activity on tRNA substrates at the non-permissive temperature...

..this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show ..

..the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli ams(ts) strain (originally defined by a prolonged bulk mRNA half-life) and in the me(ts) strain (originally defined by ..

..Thus, RhlE can replace the function of RhlB in vitro. The results in the accompanying article show that CsdA can also replace RhlB in vitro. Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays...

..in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme...

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..The product of the ams/rne/hmp1 gene, which is required for RNase E activity, was overexpressed, purified to near homogeneity by electroelution ..

..The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required...

..Moreover, decay of a model mRNA can be reconstituted in vitro by a small number of purified components in a process which is more dynamic and ATP-dependent than previously imagined...

The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E...

..Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP...

..We demonstrate here that the two rne-dependent cleavages, on both sides of the transcription terminator, are catalysed by RNase E in vitro and that the ..

..Although mRNA degradation was suspected to increase the efficiency of translational control based on several considerations, our results indicate that inhibition of mRNA degradation has no effect on the level of repression by ThrRS...

..This is the first instance in which the glycolytic flux has been shown to affect the expression of a specific gene through mRNA stability...

..By changing gene location and incorporating secondary structures and RNase E sites the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 100-fold and 750-fold, respectively...

..Host RNase E activity is responsible for the transformation defect because introduction of an rne-1 allele into rho026 cells suppressed this defect, indicating that RNAI instability due to RNase E is aggravated in ..

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..Finally, the removal of as little as seven nucleotides from the 5' end of 8-S RNA rendered it almost completely unsuitable as a substrate for RNase E...

..Therefore, both pathways require the final 3' trimming for complete processing. The endonucleolytic generation of +378/+379 RNA by pathway II was blocked by the rne-3071 mutation, suggesting that this step is carried out by RNase E.

..The mechanism that links mRNA synthesis to mRNA decay is not known...

..Insertion of a 12-15 residue 'ectopic' RNase E cleavage site from either the rne leader or 9S pre-rRNA into the 5'-non-coding region of the rpsT mRNA significantly reduces the stabilizing effect ..

..Studies of SgrS in particular are important since SgrS is the first member of a novel class of bifunctional sRNA regulators and therefore serves as a model for other similar sRNAs that will be identified in the future. ..