Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonuclease E is an essential hydrolytic endonuclease in Escherichia coli, and it plays a central role in maintaining the balance and composition of the messenger RNA population. The enzyme is also required for rRNA and tRNA processing. We have shown earlier that the highly conserved catalytic domain of E. coli RNase E is a homotetramer [Callaghan, A. J. et al. (2003) Biochemistry 42, 13848-13855]. Here, we report that this quaternary organization requires zinc. Two protomers share a single zinc ion, and quantitative analysis indicates that each protein contributes two cysteine thiols toward the coordination of the metal. The candidate cysteines are part of a motif that is conserved in the RNase E protein family, and mutation of these residues causes the partial loss of zinc, the complete disruption of the tetramer into dimers, and effective catalytic inactivation. However, these mutations do not affect RNA binding. The tetramer can be artificially maintained by disulfide bond formation, which fully displaces the zinc but largely preserves the catalytic activity. Thus, catalytic activity does not require zinc directly but does require the quaternary structure, for which the metal is essential. We propose that the RNase E tetramer has two nonequivalent subunit interfaces, one of which is mediated by a single, tetrathiol-zinc complex, which we refer to as a "Zn-link" motif. One or both interfaces organize the active site, which is distinct from the primary site of RNA binding.
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PMID:"Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease, RNase E. 1577 93

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. 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.
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PMID:RNase E cleavage in the 5' leader of a tRNA precursor. 1608 Nov 1

Several factors at transcriptional, post-transcriptional or post-translational level determine the fate of a target protein and can severely restrict its yield. Here, we focus on the post-transcriptional regulation of the biosynthesis of the periplasmic protein, penicillin amidase (PA). The PA mRNA stability was determined under depleted RNase conditions in strains carrying single or multiple RNase deletions. Single deletion of the endonuclease RNase E yielded, as the highest, a fourfold stabilization of the PA mRNA. This effect, however, was reduced twice at post-translational level. The RNase II, generating secondary exonucleolytic cleavages in the mRNA, although not significantly influencing the PA mRNA decay, led also to an increase of the amount of mature PA. The non-proportional correlation between increased mRNA longevity and amount of active enzyme propose that the rational strategies for yield improvement must be based on a simultaneous tuning of more than one yield restricting factor.
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PMID:Effect of the increased stability of the penicillin amidase mRNA on the protein expression levels. 1613 83

The functional stability of mRNA is one of the crucial factors affecting the efficiency of cell-free protein synthesis. The importance of the stability of mRNA in the prolonged synthesis of protein molecules becomes even greater when the cell-free protein synthesis is directed by PCR-amplified DNAs, because the linear DNAs are rapidly degraded by the endogenous nucleases and, thus, the continuous generation of mRNA molecules is limited. With the aim of developing a highly efficient cell-free protein synthesis system directed by PCR products, in this study, we describe a systematic approach to enhance the stability of mRNA in cell-free extracts. First, exonuclease-mediated degradation was substantially reduced by introducing a stem-loop structure at the 3'-end of the mRNA. The endonucleolytic cleavage of the mRNA was minimized by using an S30 extract prepared from an Escherichia coli strain that is deficient in a major endonuclease (RNase E). Taken together, through the retardation of the endonucleolytic and exonucleolytic degradations of the mRNA molecules, the level of protein expression from the PCR-amplified DNA templates becomes comparable to that of conventional plasmid-based reactions. The enhanced productivity of the PCR-based cell-free protein synthesis enables the high-throughput generation of protein molecules required for many post-genomic applications.
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PMID:Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions. 1626 88

In Escherichia coli the initial step in the processing or decay of many messenger and structural RNAs is mediated by the endonuclease RNase E, which forms the core of a large RNA-catalysis machine termed the degradosome. Previous experiments have identified a protein that globally modulates RNA abundance by binding to RNase E and regulating its endonucleolytic activity. Here we report the discovery of RraB, which interacts with a different site on RNase E and interferes with cleavage of a different set of transcripts. We show that expression of RraA or RraB in vivo is accompanied by dramatic, distinct, and inhibitor-specific changes in degradosome composition--and that these are in turn associated with alterations in RNA decay and global transcript abundance profiles that are dissimilar to the profile observed during simple RNase E deficiency. Our results reveal the existence of endonuclease binding proteins that modulate the remodelling of degradosome composition in bacteria and argue that such degradosome remodelling is a mechanism for the differential regulation of RNA cleavages in E. coli.
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PMID:Differential modulation of E. coli mRNA abundance by inhibitory proteins that alter the composition of the degradosome. 1677 42

In Escherichia coli, the global regulatory protein CsrA (carbon store regulator A) binds to leader segments of target mRNAs, affecting their translation and stability. CsrA activity is regulated by two noncoding RNAs, CsrB and CsrC, which act by sequestering multiple CsrA dimers. Here, we describe a protein (CsrD) that controls the degradation of CsrB/C RNAs. The dramatic stabilization of CsrB/C RNAs in a csrD mutant altered the expression of CsrA-controlled genes in a manner predicted from the previously described Csr regulatory circuitry. A deficiency in RNase E, the primary endonuclease involved in mRNA decay, also stabilized CsrB/C, although the half-lives of other RNAs that are substrates for RNase E (rpsO, rpsT, and RyhB) were unaffected by csrD. Analysis of the decay of CsrB RNA, both in vitro and in vivo, suggested that CsrD is not a ribonuclease. Interestingly, the CsrD protein contains GGDEF and EAL domains, yet unlike typical proteins in this large superfamily, its activity in the regulation of CsrB/C decay does not involve cyclic di-GMP metabolism. The two predicted membrane-spanning regions are dispensable for CsrD activity, while HAMP-like, GGDEF, and EAL domains are required. Thus, these studies demonstrate a novel process for the selective targeting of RNA molecules for degradation by RNase E and a novel function for a GGDEF-EAL protein.
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PMID:Identification of a novel regulatory protein (CsrD) that targets the global regulatory RNAs CsrB and CsrC for degradation by RNase E. 1698 May 88

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.
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PMID:Genetic analysis of polynucleotide phosphorylase structure and functions. 1708 1

Accumulation of non-metabolizable glucose-phosphate in Escherichia coli is growth inhibitory and induces a specific stress response. This is sensed and coordinated by a transcription factor SgrR that in turn activates expression of the primary effector of the stress response, a small regulatory RNA, SgrS. This RNA negatively regulates the translation and stability of the ptsG mRNA, which encodes the major glucose transporter of E. coli. The effect of SgrS on ptsG mRNA occurs through a base-pairing mechanism facilitated by the RNA chaperone Hfq. Other host factors required for the regulation by SgrS include the endonuclease RNase E and components of the RNA degradosome, particularly enolase, a glycolytic enzyme whose role in RNA degradation is currently not understood. There are many unanswered questions regarding the physiology of glucose-phosphate stress, including the cellular signals and targets involved. However, it is clear that the small RNA SgrS is required for adaptation to stress. The current model is that SgrS promotes recovery by stopping the synthesis of glucose transport proteins, which in turn limits the accumulation of toxic sugar-phosphates.
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PMID:Physiological consequences of small RNA-mediated regulation of glucose-phosphate stress. 1738 24

Mobile genetic elements have the potential to influence the expression of genes surrounding their insertion site upon invasion of a genome. Here, we examine the transcriptional organization of a ribonucleotide reductase operon (nrd) that has been invaded by an HNH family homing endonuclease, mobE. In Aeromonas hydrophila phage Aeh1, mobE has inserted into the large-subunit gene (nrdA) of aerobic ribonucleotide reductase (RNR), splitting it into two smaller genes, nrdA-a and nrdA-b. This gene organization differs from that in phages T4, T6, RB2, RB3, RB15, and LZ7, where mobE is inserted in the nrdA-nrdB intergenic region. We present evidence that the expression of Aeh1 mobE is regulated by transcriptional, posttranscriptional, and translational controls. An Aeh1-specific late promoter drives expression of mobE, but strikingly the mobE transcript is processed internally at an RNase E-like site. We also identified a putative stem-loop structure upstream of mobE that sequesters the mobE ribosome binding site, presumably acting to down regulate MobE translation. Moreover, our transcriptional analyses indicate that the surrounding nrd genes of phage Aeh1 are expressed by a different strategy than are the corresponding phage T4 genes and that transcriptional readthrough is the only mechanism by which the promoterless Aeh1 nrdB gene is expressed. We suggest that the occurrence of multiple layers of control to limit the expression of mobE to late in the Aeh1 infection cycle is an adaptation of Aeh1 to reduce any effects on expression of the surrounding nrd genes early in phage infection when RNR function is critical.
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PMID:Multiple controls regulate the expression of mobE, an HNH homing endonuclease gene embedded within a ribonucleotide reductase gene of phage Aeh1. 1744 12

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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PMID:Initiation of RNA decay in Escherichia coli by 5' pyrophosphate removal. 1761 92


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