The (6) found that during some steps of RNase E purification, the specific activity decreased and that this decrease could be reverted by adding less purified material to the purified fraction. Additionally, RNase E was found to cosediment with a high molecular weight cell membrane fraction (7). Recently, it has been shown that RNase E forms a large complex in vivo with polynucleotide phosphorylase, a 3' exoribonuclease, and other proteins (8,9) and that polynucleotide phosphorylase interacts functionally with RNase E to affect its activity (10). Additionally, GroEL, a chaperonin and heat shock protein, has been reported to copurify with an RNase E-like activity (11).Here Results) in the absence of induction of T7 RNA polymerase. At an OD60o of 0.6-1.0 unit, rifampicin was added to 100 ,tg/ml (we obtained similar results without the addition of rifampicin), and overexpression of Rne was induced by a temperature shift to 42°C for 25 min, and we did not use a longer period of induction since it resulted in a reduction of the intracellular concentration of the enzyme, possibly because of its ability to autoregulate its own synthesis (18,19). In addition, we did not observe overexpression of either enzyme using the two plasmid system when strains were tOn leave from:

The Escherichia coli RNA chaperone Hfq was discovered originally as an accessory factor of the phage Q␤ replicase. More recent work suggested a role of Hfq in cellular physiology through its interaction with ompA mRNA and small RNAs (sRNAs), some of which are involved in translational regulation. Despite their stability under certain conditions, E. coli sRNAs contain putative RNase E recognition sites, that is, A/U-rich sequences and adjacent stem-loop structures. We show herein that an RNase E cleavage site coincides with the Hfq-binding site in the 5-untranslated region of E. coli ompA mRNA as well as with that in the sRNA, DsrA. Likewise, Hfq protects RyhB RNA from in vitro cleavage by RNase E. These in vitro data are supported by the increased abundance of DsrA and RyhB sRNAs in an RNase E mutant strain as well as by their decreased stability in a hfq − strain. 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.

The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. 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.

Escherichia coli RNase E, an essential singlestranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in Synechocystis sp. and other bacteria. Furthermore, we demonstrate that the Synechocystis sp. homologue binds RNase E substrates and cleaves them at the same position as the E. coli enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to E. coli and its close relatives. We also show that the C-terminal half of E. coli RNase E is both sufficient and necessary for its physical interaction with the 3-5 exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a ''degradosome'' complex. Interestingly, however, the sequence of the C-terminal half of E. coli RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the Synechocystis sp. RNase E homologue does not function as a platform for assembly of E. coli degradosome components.E. coli RNase E is a site-specific endoribonuclease (for review, see ref. 1) that was originally identified as an activity essential for cell viability and the generation of 5S rRNA from 9S RNA, a larger precursor (2). The endoribonucleolytic activity of this enzyme is now known also to have an important role in the degradation of a number of mRNAs (for reviews, see refs. 3 and 4) and antisense RNAs that control the replication of ColE1-type and IncFII plasmids (5-7). Very recently, RNase E has been reported to shorten 3Ј poly(A) tails (8), which also are involved in determining E. coli RNA stability (for review, see ref. 9), suggesting another mechanism by which this enzyme can exercise control over RNA decay.In E. coli, RNase E is a component of the degradosome, a multiprotein complex whose other major components are polynucleotide phosphorylase (PNPase), a 3Ј-5Ј exoribonuclease, the RhlB RNA helicase, and the glycolytic enzyme enolase (10-13). Three minor components of the degradosome, DnaK,

SummaryThe Escherichia coli Sm-like host factor I (Hfq) is thought to play direct and indirect roles in posttranscriptional regulation by targeting small regulatory RNAs and mRNAs. In this study, we have used proteomics to identify new mRNA targets of Hfq. We have identified 11 candidate proteins, synthesis of which was differentially affected in a hfq -background. The effect of Hfq on some of the corresponding mRNAs including fur , gapA , metF , ppiB and sodB mRNA was assessed, using different in vitro and in vivo methods. This allowed us to distinguish between direct and indirect effects of Hfq in modulating the translational activities of these mRNAs. From the collection of mRNAs tested, only fur and sodB mRNA, encoding the master regulator of iron metabolism and the iron superoxide dismutase, respectively, were found to be regulated by Hfq. Fur is known to be a negative regulator of transcription of the small RNA RyhB. Mutations in the sodB leader and compensating mutations in RyhB revealed that RyhB in turn represses translation of sodB mRNA, explaining the previously reported positive control of sodB by Fur. These data assign a role to Hfq in regulation of iron uptake and in switching off of iron scavenger genes.

Studies in pro- and eukaryotes have revealed that translation can determine the stability of a given messenger RNA. In bacteria, intrinsic mRNA signals can confer efficient ribosome binding, whereas translational feedback inhibition or environmental cues can interfere with this process. Such regulatory mechanisms are often controlled by RNA-binding proteins, small noncoding RNAs and structural rearrangements within the 5' untranslated region. Here, we review molecular events occurring in the 5' untranslated region of primarily Escherichia coli mRNAs with regard to their effects on mRNA stability.

The intricate regulation of the Escherichia coli rpoS gene, which encodes the stationary phase sigma-factor s S , includes translational activation by the noncoding RNA DsrA. We observed that the stability of rpoS mRNA, and concomitantly the concentration of s S , were significantly higher in an RNase III-deficient mutant. As no decay intermediates corresponding to the in vitro mapped RNase III cleavage site in the rpoS leader could be detected in vivo, the initial RNase III cleavage appears to be decisive for the observed rapid inactivation of rpoS mRNA. In contrast, we show that base-pairing of DsrA with the rpoS leader creates an alternative RNase III cleavage site within the rpoS/DsrA duplex. This study provides new insights into regulation by small regulatory RNAs in that the molecular function of DsrA not only facilitates ribosome loading on rpoS mRNA, but additionally involves an alternative processing of the target.

The trimeric translation initiation factor a/eIF2 of the crenarchaeon Sulfolobus solfataricus is pivotal for binding of initiator tRNA to the ribosome. Here, we present in vitro and in vivo evidence that the a/eIF2 ␥-subunit exhibits an additional function with resemblance to the eukaryotic cap-complex. It binds to the 5-triphosphate end of mRNA and protects the 5 part from degradation. This unprecedented capacity of the archaeal initiation factor further indicates that 5 3 3 directional mRNA decay is a pathway common to all domains of life. In Escherichia coli, the decay of most RNA transcripts appears to be initiated by 5Ј pyrophosphate removal (1), followed by endonucleolytic cleavages that are generated by RNase E (2, 3). The intermediate cleavage products are further degraded by the 3Ј 3 5Ј exonucleases polynucleotide phosphorylase (PNPase), RNase II, and oligoribonuclease, converting the decay intermediates into poly-and mononucleotides (4). At variance with E. coli, Bacillus subtilis possesses a 5Ј 3 3Ј exonuclease activity, which could explain why RNAs in this organism are stabilized for great distances downstream of stable secondary structures or bound ribosomes (5). In eukaryotes, mRNA decay is mainly catalyzed by exonucleases (6). Eukaryotic mRNAs generally have a 7-methylguanosine cap at their 5Ј end and a poly(A) tail at their 3Ј end. Removal of these terminal modifications is considered rate-limiting for mRNA decay (7). Translation initiation factor eIF4E binds to the 7-methylguanosine cap and thereby protects the cap structure from the decapping enzyme and consequently the mRNA from 5Ј 3 3Ј exonucleolytic decay (7). Different RNases with either endo-or exonuclease activity (8-11) have been inferred or described in Archaea. In S. solfataricus a 3Ј 3 5Ј directional decay by a multisubunit exosome complex has been demonstrated in vitro (10). In addition, the Sulfolobus solfataricus exosome is able to polyadenylate the 3Ј end of RNAs in the presence of ADP (12). With the exception of the exosome, no other endo-or exonuclease activities have been described in S. solfataricus. At variance with a previous study wherein the longevity of selected S. solfataricus mRNAs was found to be rather high (13), a recent microarraybased analysis (14) indicated a rather short mRNA half-life, comparable with that of bacterial mRNAs.Two different mechanisms for translational initiation seem to exist in Sulfolobus (15). One is based on a canonical SD/anti-SD interaction and operates on internal cistrons of polycistronic mRNAs. In contrast, monocistronic mRNAs and proximal genes of polycistronic mRNAs are frequently devoid of a 5Ј untranslated region. Decoding of these leaderless mRNAs requires, analogously to Bacteria (16), pairing of the start codon with initiator-tRNA (15). The complexity of archaeal translational initiation seems to be underscored by the presence of a largerthan-bacterial set of factors because Archaea encode Ϸ10 orthologs of eukaryal and bacterial initiation factors (17). Like its eukaryotic counterp...