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Query: UNIPROT:P06889 (Mol)
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Processing of 9 S precursor RNA in Escherichia coli requires the endoribonuclease RNase E, which makes two cleavages to liberate p5, the immature form of 5 S rRNA. The contributions of primary and secondary structure to RNase E-mediated cleavage of 9 S RNA were investigated. The structure of the 5' domain of 9 S RNA was probed by partial ribonuclease digestion and chemical modification. Our structural analysis of 9 S RNA supports a model in which the 5' spacer domain folds into tandem hairpins so that the first processing cleavage site 5' to the 5 S moiety resides in a stretch of single-stranded residues. Site-directed mutagenesis of a cloned 9 S RNA sequence was performed and synthetic transcripts derived from a variety of such mutant templates were assayed as substrates for RNase E-dependent endonuclease activity in fractionated extracts. Partial or complete deletion of the 5 S sequence did not eliminate site-specific processing of 9 S RNA. Mutations affecting the 5' domain revealed that secondary structure upstream from the first cleavage site is important in maintaining efficient processing. However, secondary structure downstream from either cleavage site is dispensable. Our results suggest that RNase E specifically recognizes and cleaves single-stranded RNA sequences only when presented in a proper conformational context. Adjacent secondary structures appear to play a direct and critical role in the enzyme's recognition of its substrate. Additionally, it may serve to anchor single-stranded regions to ensure the availability of the RNase E cleavage sites.
J Mol Biol 1992 Dec 20
PMID:Structural requirements for the processing of Escherichia coli 5 S ribosomal RNA by RNase E in vitro. 147 79

We have constructed a collection of Escherichia coli strains which differ by point mutations in the ribosome binding site (RBS) that drives the translation of the lacZ gene. These mutations affect the Shine-Dalgarno sequence or the initiation codon, or create secondary structures that sequester these elements, and result in a 200-fold variation in beta-galactosidase expression. Surprisingly, these variations of expression are paralleled by nearly equivalent changes in the lacZ mRNA level. The ratio of the beta-galactosidase expression to the mRNA level reflects the average spacing between translating ribosomes: hence, paradoxically, mutations that affect translation initiation do not correspondingly change this spacing. Further analysis of the mRNA level variations shows that they originate from two independent mechanisms. When beta-galactosidase expression exceeds a threshold corresponding roughly to one translation event per transcript, the variations in the efficiency of translation initiation affect largely the chemical and functional lifetimes of the mRNA. We further show that the rate-limiting step in the chemical decay process is an RNase E-dependent cleavage, which is outcompeted by translation initiation. Below this expression threshold, the mRNA lifetime levels out and strain-to-strain variations in mRNA level arise solely from polarity effects. We suggest that, in this activity range, most mRNA molecules that escape polarity are crossed by a single ribosome, and hence are identical from the viewpoint of degradation. Altogether, the tight couplings between translation initiation on one hand, polarity and/or mRNA degradation on the other, result in translation initiation events being closely spaced in time even from inefficient RBS, at the expense of the mRNA level. Finally, we evocate the possible beneficial consequences of a coupling between translation, transcription and mRNA degradation, for the management of cellular resources.
J Mol Biol 1992 Aug 05
PMID:Interdependence of translation, transcription and mRNA degradation in the lacZ gene. 150 17

We have established that the long non-coding intercistronic region of the dicB operon of Escherichia coli expresses a trans-acting division inhibitor specified by a region dicF, at most 65 nucleotides-long. The present study deals with the processing of dicBF operon mRNA in vivo, and identifies the dicF gene product as a 53 nucleotide RNA species. A sequence at the end of DicF resembles, and behaves as, a Rho-independent terminator, but further processing of readthrough transcripts, presumably by RNase III, followed by a limited 3' to 5' degradation, appears to generate additional DicF-RNA 3' ends. For the 5' end of DicF-RNA, our results show that a 190 nucleotide precursor DicF-RNA species is formed by cleavage at an RNase III site, while the 53 nucleotide minimal DicF-RNA is generated by further processing requiring the presence of an active form of RNase E in vivo. These data indicate that an untranslated product derived from an operon RNA can have a regulatory activity by affecting cell division.
J Mol Biol 1990 Apr 05
PMID:Escherichia coli cell division inhibitor DicF-RNA of the dicB operon. Evidence for its generation in vivo by transcription termination and by RNase III and RNase E-dependent processing. 169 Dec 99

The transcripts of the rpsO-pnp operon of Escherichia coli, coding for ribosomal protein S15 and polynucleotide phosphorylase, are processed at four sites in the 249 nucleotides of the intercistronic region. The initial processing step in the decay of the pnp mRNA is made by RNase III, which cuts at two sites upstream from the pnp gene. The other two cleavages are dependent on the wild-type allele of the rne gene, which encodes the endonucleolytic enzyme RNase E. The cuts are made 37 nucleotides apart at the base of the stem-loop structure of the rho-independent attenuator located downstream from rpsO. The cleavage downstream from the attenuator generates an rpsO mRNA.nearly identical with the monocistronic attenuated transcript, while the cleavage upstream from the transcription attenuator gives rise to an rpsO mesage lacking the terminal 3' hairpin structure. The rapid degradation of the processed mRNA in an rne+ strain, compared to the slow degradation of the transcript that accumulates in an rne- strain, suggests that RNase E initiates the decay of the rpsO message by removing the stabilizing stem-loop at the 3' end of the RNA.
J Mol Biol 1991 Jan 20
PMID:Decay of mRNA encoding ribosomal protein S15 of Escherichia coli is initiated by an RNase E-dependent endonucleolytic cleavage that removes the 3' stabilizing stem and loop structure. 170 67

Escherichia coli RNase E is known to process RNA precursors at specific sites. We show that this endoribonuclease has a general role in E. coli mRNA turnover and affects the stability of specific transcripts. The effect of the rne mutation on functional stability of mRNA was much less pronounced than that on chemical stability, although the expression of some genes was affected. The E. coli ams (altered mRNA stability) mutation was found to have phenotypes indistinguishable from those of the rne mutation, affecting both 9S RNA and T4 gene 32 mRNA processing. The rne and ams mutations were both complemented by the same 3.7 kb fragment of E. coli DNA and are probably allelic. RNase E is the first endoribonuclease identified as having a general role in the chemical decay of E. coli mRNA.
Mol Microbiol 1990 Dec
PMID:RNase E, an endoribonuclease, has a general role in the chemical decay of Escherichia coli mRNA: evidence that rne and ams are the same genetic locus. 170 38

A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by 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 that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical.
Mol Microbiol 1991 Apr
PMID:The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene. 171 82

We show in 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 aberrant 9S RNA processing). The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8 kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the ams(ts) and rne(ts) strains. This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Ams(ts) and the Rne(ts) phenotype with regard to the ompA cleavages. In addition we showed that the ams(ts) strain suppresses 9S RNA processing to 5S RNA to the same extent as the rne(ts) strain, and that the rne(ts0 strain has a prolonged bulk mRNA half-life, as was reported for the ams(ts) strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K.
Mol Microbiol 1991 Apr
PMID:Genetic studies of cleavage-initiated mRNA decay and processing of ribosomal 9S RNA show that the Escherichia coli ams and rne loci are the same. 171 83

Using T7 RNA polymerase and specific constructs derived from 5S rRNA and RNA I genes, we generated substrates for the RNA processing enzyme RNase E. Using these substrates we have shown that a 3.2 kb DNA fragment that complements the rne-3071 mutation can express RNase E activity. We also found that T7 RNA polymerase terminates within the 5S rRNA gene.
Mol Gen Genet 1991 Aug
PMID:The rne gene is the structural gene for the processing endoribonuclease RNase E of Escherichia coli. 171 77

Cells overexpressing the RNA-processing enzymes RNase III, RNase E and RNase P were fractionated into membrane and cytoplasm. The RNA-processing enzymes were associated with the membrane fraction. The membrane was further separated to inner and outer membrane and the three RNA-processing enzymes were found in the inner membrane fraction. By assaying for these enzymatic activities we showed that even in a normal wild-type strain of Escherichia coli these enzymes fractionate primarily with the membrane. The RNA part of RNase P is found in the cytosolic fraction of cells overexpressing this RNA, while the overexpressed RNase P protein sediments with the membrane fraction; this suggests that the RNase P protein anchors the RNA catalytic moiety of the enzyme to a larger entity. The implications of these findings for the cellular organization of the RNA-processing enzymes in the cell are discussed.
Mol Microbiol 1991 Jul
PMID:Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Escherichia coli cell. 194 11

We have analysed transcription and mRNA processing for the gene 32 region of five phages related to T4. Two different organizations of gene 32 proximal promoters were found. In T4 and M1, middle- and late-mode promoters are separated by 50 nucleotides and located within an upstream open reading frame. In T2, K3, Ac3, and Ox2, the 626bp T4 sequence that includes these promoters is replaced by a 59bp sequence containing overlapping middle and late promoters. The RNase E-dependent processing of the g32 mRNAs is conserved in all of the phages. The processing site immediately upstream of g32 in T4 and M1 has been replaced in the other phages by a different sequence that is also cleaved by RNase E. The remarkable conservation of these regulatory features, despite the sequence divergences, suggests that they play an important role in the control of gene expression.
Mol Microbiol 1991 Mar
PMID:Gene 32 transcription and mRNA processing in T4-related bacteriophages. 204 53


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