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At least 919 records · Page 51Linked to original sources
PubMed1996

Polynucleotide phosphorylase is required for the rapid degradation of the RNase E-processed rpsO mRNA of Escherichia coli devoid of its 3' hairpin.

The monocistronic transcript of rpsO undergoes an endonucleolytic cleavage downstream of the coding sequence, which removes the hairpin of the transcription terminator and initiates the rapid degradation of the message. 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 RNase E-processed rpsO message is rapidly degraded by polynucleotide phosphorylase, while RNase II produces stable decay intermediates. Moreover, we show that RNase E cuts in vitro the coding sequence of the rpsO mRNA at several sites which are not detected in vivo.

Base Sequence↗
PubMed1997

Sok antisense RNA from plasmid R1 is functionally inactivated by RNase E and polyadenylated by poly(A) polymerase I.

The hok/sok system of plasmid R1, which mediates plasmid stabilization by the killing of plasmid-free cells, codes for two RNA species, Sok antisense RNA and hok mRNA. Sok RNA, which is unstable, inhibits translation of the stable hok mRNA. The 64nt Sok RNA folds into a single stem-loop domain with an 11 nt unstructured 5' domain. The initial recognition reaction between Sok RNA and hok mRNA takes place between the 5' domain and the complementary region in hok mRNA. In this communication we examine the metabolism of Sok antisense RNA. We find that RNase E cleaves the RNA 6nt from its 5' end and that this cleavage initiates Sok RNA decay. The RNase E cleavage occurs in the part of Sok RNA that is responsible for the initial recognition of the target loop in hok mRNA and thus leads to functional inactivation of the antisense. The major RNase E cleavage product (denoted pSok-6) is rapidly degraded by polynucleotide phosphorylase (PNPase). Thus, the RNase E cleavage tags pSok-6 for further rapid degradation by PNPase from its 3' end. 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.

Bacterial Proteins↗
PubMed2000

Endoribonuclease RNase III is essential in Bacillus subtilis.

The rncS gene of Bacillus subtilis encodes Bs-RNase III, a narrow-specificity endoribonuclease. Previous attempts to disrupt rncS were unsuccessful. Here, a strain was constructed in which Bs-RNase III expression was dependent upon transcription of rncS from a temperature-sensitive plasmid. Growth of this strain at the non-permissive temperature resulted in 90-95% cell death, and virtually all the cells that survived retained the rncS-expressing plasmid. Thus, we conclude that rncS is essential in B. subtilis. The rncS conditional strain also revealed that Bs-RNase III participates in the processing of ribosomal RNA, in addition to processing small cytoplasmic RNA, a member of the signal recognition particle RNA family. Most significantly, a rare rncS null strain was isolated that will aid further study of the critical role Bs-RNase III plays in B. subtilis.

Bacillus subtilis↗
PubMed2000

Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly.

We demonstrate here that the assembly of the RNase E-based degradosome of Escherichia coli is not required for normal mRNA decay in vivo. In contrast, deletion of the arginine-rich RNA binding site (ARRBS) from the RNase E protein slightly impairs mRNA decay. When both the degradosome scaffold region and the ARRBS are missing, mRNA decay is dramatically slowed, but 9S rRNA processing is almost normal. An extensive RNase E truncation mutation (rnedelta610) had a more pronounced mRNA decay defect at 37 degrees C than the temperature-sensitive rne-1 allele at 44 degrees C. Taken together, these data suggest that the inviability associated with inactivation of RNase E is not related to defects in either mRNA decay or rRNA processing.

Endoribonucleases↗
PubMed2001

Bacteriophage T7 protein kinase phosphorylates RNase E and stabilizes mRNAs synthesized by T7 RNA polymerase.

The T7 protein encoded by the early gene 0.7 exhibits bifunctional activity. Whereas its C-terminal one-third participates in host transcription shut-off, the N-terminal two-thirds bears a protein kinase ('PK') activity that can phosphorylate a number of host proteins in addition to itself. Here, we show that, when PK is expressed in uninfected Escherichia coli cells, the C-terminal half of RNase E and the associated RNA helicase RhlB are heavily phosphorylated. Meanwhile, a subset of RNase E substrates, including the lac and cat mRNAs synthesized by bacteriophage T7 RNA polymerase (RNAP), are stabilized. These mRNAs are genuinely less stable than their counterparts synthesized by E. coli RNAP, because T7 RNAP outpaces translating ribosomes, creating naked, RNase E-sensitive mRNA stretches behind itself. Thus, PK alleviates this effect of desynchronizing transcription and translation. The relationship between the modification of RNase E and RhlB and these mRNA stabilization effects, which may be relevant to the stability of late T7 mRNAs during infection, is discussed.

DNA-Directed RNA Polymerases↗
PubMed2002

RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters.

The rne gene of Escherichia coli encodes RNase E, an essential endoribonuclease that is involved in both mRNA decay and rRNA processing. Here we present evidence that the gene is transcribed from three promoters: p1, p2 and p3. The p2 and p3 promoters map 34 and 145 nt upstream from the previously characterized rne promoter, p1, generating unusually long 5' UTRs of 395 and 506 nt respectively. Based on promoter-lacZ transcriptional fusions, p1 is a more efficient promoter than either p2 or p3. Low copy number or single copy number vectors carrying rne transcribed from either p1, p2 or p3 alone complement the rne 1018::bla deletion mutation at 30 degrees C, 37 degrees C and 44 degrees C. However, normal autoregulation requires the presence of all three promoters. A comparison among intracellular levels of RNase E, the half-lives of the rpsO, rpsT and rne mRNAs, and growth rates, indicates that the cell contains a considerable excess of RNase E protein. In addition, when the rne transcript is stabilized at low RNase E levels, it is not efficiently translated.

5' Untranslated Regions↗
PubMed2002

Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase E.

Polyadenylation in Escherichia coli has been implicated in the destabilization of a variety of transcripts. 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. Here, we show that the half-lives of both the pnp and rne transcripts are dependent on the intracellular level of polyadenylated transcripts. In addition, experiments using pnp-lacZ and rne-lacZ translational fusions demonstrate that the variations in transcript stability and protein levels arise from alterations in the autoregulation of both genes. Further support for this conclusion is provided by the fact that, in an rne mutant in which autoregulation is inactivated by deletion of most of the 5' untranslated region, variations in the level of polyadenylated transcripts no longer affect RNase E protein expression. Of even more interest is the fact that the presence of a functional degradosome is essential for RNase E to detect increased levels of poly(A). Thus, it appears that polyadenylation of transcripts in E. coli serves as a sensing mechanism by which the cell adjusts the levels of both RNase E and PNPase.

5' Untranslated Regions↗
PubMed2002

Function in Escherichia coli of the non-catalytic part of RNase E: role in the degradation of ribosome-free mRNA.

RNase E contains a large non-catalytic region that binds RNA and the protein components of the Escherichia coli RNA degradosome. The rne gene was replaced with alleles encoding deletions in the non-catalytic part of RNase E. All the proteins are stable in vivo. RNase E activity was tested using a P(T7)-lacZ reporter gene, the message of which is particularly sensitive to degradation because translation is uncoupled from transcription. The non-catalytic region has positive and negative effectors of mRNA degradation. Disrupting RhlB and enolase binding resulted in hypoactivity, whereas disrupting PNPase binding resulted in hyperactivity. Expression of the mutant proteins in vivo anticorrelates with activity showing that autoregulation compensates for defective function. There is no simple correlation between RNA binding and activity in vivo. An allele (rne131), expressing the catalytic domain alone, was put under P(lac) control. In contrast to rne+,low expression of rne131 severely affects growth. Even with autoregulation, all the mutants are less fit when grown in competition with wild type. Although the catalytic domain of RNase E is sufficient for viability, our work demonstrates that elements in the non-catalytic part are necessary for normal activity in vivo.

Endoribonucleases↗
PubMed2003

Ectopic RNase E sites promote bypass of 5'-end-dependent mRNA decay in Escherichia coli.

In Escherichia coli, 5'-terminal stem-loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5'-terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two-thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. 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 of the terminal stem-loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5'-terminus, and exploit an alternative 'internal entry' pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5'-termini, the use of internal entry for bypass of barriers to decay, 'ectopic sites' and the role of translating ribosomes.

Codon↗
PubMed2002

Local and systemic wound-induction of RNase and nuclease activities in Arabidopsis: RNS1 as a marker for a JA-independent systemic signaling pathway.

Induction of defense-related genes is one way in which plants respond to mechanical injury. We investigated whether RNases are involved in the wound response in Arabidopsis thaliana. As in other plant systems, several activities are induced with various timings in damaged leaves, stems and seedlings in Arabidopsis, including at least three bifunctional nucleases, capable of degrading both RNA and DNA, as well as RNS1, a member of the ubiquitous RNase T(2) family of RNases. The strong induction of RNS1 is particularly interesting because it occurs both locally and systemically following wounding. The systemic induction of this RNase indicates that members of this family may be involved in defense mechanisms in addition to their previously hypothesized functions in nutrient recycling and remobilization. Additionally, the systemic induction appears to be controlled independently of jasmonic acid, and the local induction of RNS1 and the nuclease activities are independent of both JA and oligosaccharide elicitors. Consequently, a novel systemic pathway, likely involving a third signal, appears to exist in Arabidopsis.

Arabidopsis↗
PubMed2000

Homodimeric reverse transcriptases from rous sarcoma virus mutated within the polymerase or RNase H active site of one subunit are active.

Heterodimeric reverse transcriptase (RT) alphabeta from Rous sarcoma virus (RSV) possesses an asymmetric subunit organization with the polymerase and RNase H active sites localized in the alpha subunit. To determine whether homodimeric RSV RTs alpha (63 kDa) or beta (95 kDa) assume alpha subunit organization similar to that of the heterodimer, an essential aspartic acid residue was mutated in the active site of either the polymerase (Asp181 > Asn) or the RNase H (Asp505 > Asn). Homodimeric alpha or beta RT consisting of one wild-type and one mutated subunit exhibit polymerase or RNase H activity, respectively, whereas the corresponding doubly mutated enzymes are inactive, indicating that the catalytic sites of the polymerase and RNase H domains are formed by only one subunit of the homodimer.

Animals↗
PubMed2001

Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.

Animals↗
PubMed2001

Semiquantitative polymerase chain reaction in RNase-producing tissues: Analysis of the developing pancreas.

BACKGROUND/PURPOSE: Many studies in pediatric surgical research use a quantitative analysis of gene expression in microscopic quantities of tissue. The authors describe an analysis of the beta-tubulin mRNA content of the embryonic pancreas, which contains abundant endogenous RNases. A detailed analysis of this RNase-containing system will provide a good template for analysis of other potentially simpler systems. METHODS: Embryonic mouse pancreases were harvested at serial gestational ages. DAPI nuclear staining allowed for counting of cells. cDNA was amplified using a fluoresceinated primer and the normalized fluorescence determined. Known numbers of molecules were amplified in parallel as a standard control. RESULTS: The number of cells increased from 38,000 to 2,700,000 between embryonic day 10.5 (E10.5) and E18.5. mRNA for beta-tubulin did not increase proportionately. Assuming a yield of 100% at E10.5 when no RNases are present, the yield of expected mRNA was 65.3% at E12.5, 13.8% at E15.5, and 0.9% at E18.5, presumably because of the appearance of RNases. CONCLUSIONS: Several parameters must be considered in performing semiquantitative reverse transcription polymerase chain reaction: (1) the yield of RNA based on the projected amount of mRNA, (2) the number of cells in the tissue, and (3) a known number of template molecules amplified in parallel.

Animals↗
PubMed2003

Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P.

The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited.

Base Sequence↗
PubMed2005

Evidence in vivo that the DEAD-box RNA helicase RhlB facilitates the degradation of ribosome-free mRNA by RNase E.

The RNA degradosome of Escherichia coli is a ribonucleolytic multienzyme complex containing RNase E, polynucleotide phosphorylase, RhlB, and enolase. Previous in vitro and in vivo work has shown that RhlB facilitates the exonucleolytic degradation of structured mRNA decay intermediates by polynucleotide phosphorylase in an ATPase-dependent reaction. Here, we show that deleting the gene encoding RhlB stabilizes a lacZ mRNA transcribed by bacteriophage T7 RNA polymerase. Deleting the gene encoding enolase has little if any effect. Other messages transcribed by T7 polymerase are also stabilized by DeltarhlB. The effect of point mutations inactivating RhlB is comparable with the effect of deleting the gene. Primer extension analysis of the lacZ message indicates that RhlB facilitates endoribonucleolytic cleavage by RNase E, demonstrating a functional interaction between the RNA helicase and the endoribonuclease. The possible physiological role of an RhlB-RNase E pathway and the mechanisms by which RhlB could facilitate RNase E cleavage are discussed.

Blotting, Western↗
PubMed2006

Structure of Pfu Pop5, an archaeal RNase P protein.

We have used NMR spectroscopy and x-ray crystallography to determine the three-dimensional structure of PF1378 (Pfu Pop5), one of four protein subunits of archaeal RNase P that shares a homolog in the eukaryotic enzyme. RNase P is an essential and ubiquitous ribonucleoprotein enzyme required for maturation of tRNA. In bacteria, the enzyme's RNA subunit is responsible for cleaving the single-stranded 5' leader sequence of precursor tRNA molecules (pre-tRNA), whereas the protein subunit assists in substrate binding. Although in bacteria the RNase P holoenzyme consists of one large catalytic RNA and one small protein subunit, in archaea and eukarya the enzyme contains several (> or =4) protein subunits, each of which lacks sequence similarity to the bacterial protein. The functional role of the proteins is poorly understood, as is the increased complexity in comparison to the bacterial enzyme. Pfu Pop5 has been directly implicated in catalysis by the observation that it pairs with PF1914 (Pfu Rpp30) to functionally reconstitute the catalytic domain of the RNA subunit. The protein adopts an alpha-beta sandwich fold highly homologous to the single-stranded RNA binding RRM domain. Furthermore, the three-dimensional arrangement of Pfu Pop5's structural elements is remarkably similar to that of the bacterial protein subunit. NMR spectra have been used to map the interaction of Pop5 with Pfu Rpp30. The data presented permit tantalizing hypotheses regarding the role of this protein subunit shared by archaeal and eukaryotic RNase P.

Amino Acid Sequence↗
PubMed2000

Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection.

The mammalian RNase A superfamily comprises a diverse array of ribonucleolytic proteins that have a variety of biochemical activities and physiological functions. Two rapidly evolving RNases of higher primates are of particular interest as they are major secretory proteins of eosinophilic leukocytes and have been found to possess anti-pathogen activities in vitro. To understand how these RNases acquired this function during evolution and to develop animal models for the study of their functions in vivo, it is necessary to investigate these genes in many species. Here, we report the sequences of 38 functional genes and 23 pseudogenes of the eosinophil-associated RNase (EAR) family from 5 rodent species. Our phylogenetic analysis of these genes showed a clear pattern of evolution by a rapid birth-and-death process and gene sorting, a process characterized by rapid gene duplication and deactivation occurring differentially among lineages. This process ultimately generates distinct or only partially overlapping inventories of the genes, even in closely related species. Positive Darwinian selection also contributed to the diversification of these EAR genes. The striking similarity between the evolutionary patterns of the EAR genes and those of the major histocompatibility complex, immunoglobulin, and T cell receptor genes stands in strong support of the hypothesis that host-defense and generation of diversity are among the primary physiological function of the rodent EARs. The discovery of a large number of divergent EARs suggests the intriguing possibility that these proteins have been specifically tailored to fight against distinct rodent pathogens.

Amino Acid Sequence↗
PubMed2003

An unusual mechanism of bacterial gene expression revealed for the RNase P protein of Thermus strains.

The RNase P protein gene (rnpA) completely overlaps the rpmH gene (encoding ribosomal protein L34) out of frame in the thermophilic bacterium Thermus thermophilus. This results in the synthesis of an extended RNase P protein (C5) of 163 aa and, by inference, of 240 aa in the related strain Thermus filiformis. Start codons of rnpA and rpmH, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between L34 and C5 translation and, accordingly, between ribosome and RNase P biosynthesis. Within the sequence encoding the N-terminal extensions and downstream of rpmH, several Thermus species exhibit in-frame deletionsinsertions, suggesting relaxed constraints for sequence conservation in this region. Roughly the N-terminal third of T. thermophilus C5 was further shown to be dispensable for RNase P function in vitro by using a precursor tRNA(Gly) substrate from the same organism. Taken together, these data reveal a mode of gene expression that is to our knowledge unprecedented in bacteria.

Amino Acid Sequence↗