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:3.1.13.1 (exoribonuclease)
732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.
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PMID:RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells. 164 89

We have altered by site-directed mutagenesis the 3' terminal adenosine residue of a tRNA(Tyrsu3+) gene encoded on a single-copy plasmid and examined the consequences of these substitutions on suppressor activity in vivo. Our data show that mutant su3 genes containing 3'-CCC, -CCG, or -CCU termini instead of -CCA can be efficiently transcribed and processed in Escherichia coli to generate functional suppressor tRNAs. However, in contrast to normal tRNA genes, both tRNA nucleotidyltransferase and exoribonuclease activities are required to obtain suppression by the mutant tRNAs, indicating that removal of the incorrect 3' terminal residue and resynthesis of the normal -CCA terminus are occurring in this situation. In addition, a low level of suppressor activity and tRNA repair was found in cells devoid of tRNA nucleotidyltransferase, suggesting that an additional activity able to partially repair the 3' end of tRNA is present in E. coli. The use of mutant strains lacking one or several exoribonucleases revealed that the various RNAses have very different specificities for removal of incorrect 3' residues and that these differ greatly from their action on CCA-ending tRNA. These data show that the 3' terminal adenosine residue is necessary for tRNA function in vivo and that cells can compensate for its alteration by changes in the normal pathway of tRNA metabolism.
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PMID:Substitution of the 3' terminal adenosine residue of transfer RNA in vivo. 768 99

RNase PH is one of the exoribonucleases that catalyze the 3' end processing of tRNA in bacteria. RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity. In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-A resolution. RNase PH has the typical alpha/beta fold, which forms a hexameric ring structure as a trimer of dimers. This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease. Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site. Mutational analyses of these residues showed their importance in the phosphorolysis reaction. A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs.
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PMID:Crystal structure of the tRNA processing enzyme RNase PH from Aquifex aeolicus. 1274 47

RNase PH is a phosphate-dependent exoribonuclease that catalyzes the removal of nucleotides at the 3' end of the tRNA precursor, leading to the release of nucleoside diphosphate, and generates the CCA end during the maturation process. The 1.9-A crystal structures of the apo and the phosphate-bound forms of RNase PH from Pseudomonas aeruginosa reveal a monomeric RNase PH with an alpha/beta-fold tightly associated into a hexameric ring structure in the form of a trimer of dimers. A five ion pair network, Glu-63-Arg-74-Asp-116-Arg-77-Asp-118 and an ion-pair Glu-26-Arg-69 that are positioned symmetrically in the trimerization interface play critical roles in the formation of a hexameric ring. Single or double mutations of Arg-69, Arg-74, or Arg-77 in these ion pairs leads to the dissociation of the RNase PH hexamer into dimers without perturbing the overall monomeric structure. The dissociated RNase PH dimer completely lost its binding affinity and catalytic activity against a precursor tRNA. Our structural and mutational analyses of RNase PH demonstrate that the hexameric ring formation is a critical feature for the function of members of the RNase PH family.
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PMID:Probing the functional importance of the hexameric ring structure of RNase PH. 1457 94

The 3' processing of most bacterial precursor tRNAs involves exonucleolytic trimming to yield a mature CCA end. This step is carried out by RNase T, a member of the large DEDD family of exonucleases. We report the crystal structures of RNase T from Escherichia coli and Pseudomonas aeruginosa, which show that this enzyme adopts an opposing dimeric arrangement, with the catalytic DEDD residues from one monomer closely juxtaposed with a large basic patch on the other monomer. This arrangement suggests that RNase T has to be dimeric for substrate specificity, and agrees very well with prior site-directed mutagenesis studies. The dimeric architecture of RNase T is very similar to the arrangement seen in oligoribonuclease, another bacterial DEDD family exoribonuclease. The catalytic residues in these two enzymes are organized very similarly to the catalytic domain of the third DEDD family exoribonuclease in E. coli, RNase D, which is monomeric.
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PMID:Crystal structure of RNase T, an exoribonuclease involved in tRNA maturation and end turnover. 1743 14

The first step in the current model for the processing and maturation of mono- and polycistronic tRNA precursors in Escherichia coli involves initial cleavages by RNase E 1-3 nt downstream of each chromosomally encoded CCA determinant. Subsequently, each mature 5' terminus is generated by single RNase P cleavage, while the 3' terminus undergoes exonucleolytic processing by a combination of 3' --> 5' exonucleases. Here we describe for the first time a previously unidentified pathway for the maturation of tRNAs in polycistronic operons (valV valW and leuQ leuP leuV) where the processing of the primary transcripts is independent of RNase E. Rather, RNase P cleavages separate the individual tRNA precursors with the concomitant formation of their mature 5' termini. Furthermore, both polynucleotide phosphorylase (PNPase) and RNase II are required for the removal of the 3' Rho-dependent terminator sequences. Our data indicate that RNase P substrate recognition is more complex than previously envisioned.
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PMID:Ribonuclease P processes polycistronic tRNA transcripts in Escherichia coli independent of ribonuclease E. 1798 36

Enzymes from several gene families modify RNA molecules at their extremities. These reactions occur in several cellular compartments and affect every class of RNA. To assess the diversity of a subclass of these enzymes, we searched Chlamydomonas for open reading frames (ORFs) potentially encoding exoribonucleases, poly(A) polymerases, and proteins known to associate with and/or regulate them. The ORFs were further analyzed for indications of protein localization to the nucleus, cytosol, mitochondrion, and/or chloroplast. By comparing predicted proteins with homologs in Arabidopsis and yeast, we derived several tentative conclusions regarding RNA 5'- and 3'-end metabolism in Chlamydomonas. First, the alga possesses only one each of the following likely organellar enzymes: polynucleotide phosphorylase, hydrolytic exoribonuclease, poly(A) polymerase, and CCA transferase, a surprisingly small complement. Second, although the core of the nuclear/cytosolic exosome decay complex is well conserved, neither nucleus-specific activators nor the cytosolic exosome activators are present. Finally, our discovery of nine noncanonical poly(A) polymerases, a divergent family retaining the catalytic domains of conventional poly(A) polymerases, leads to the hypothesis that polyadenylation may play an especially important regulatory role throughout the Chlamydomonas cell, stabilizing some transcripts and targeting degradation machinery to others.
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PMID:Genome-based analysis of Chlamydomonas reinhardtii exoribonucleases and poly(A) polymerases predicts unexpected organellar and exosomal features. 1849 45

Processing of the 3' terminus of tRNA in many organisms is carried out by an endoribonuclease termed RNase Z or 3'-tRNase, which cleaves after the discriminator nucleotide to allow addition of the universal -CCA sequence. In some eubacteria, such as Escherichia coli, the -CCA sequence is encoded in all known tRNA genes. Nevertheless, an RNase Z homologue (RNase BN) is still present, even though its action is not needed for tRNA maturation. To help identify which RNA molecules might be potential substrates for RNase BN, we carried out a detailed examination of its specificity and catalytic potential using a variety of synthetic substrates. We show here that RNase BN is active on both double- and single-stranded RNA but that duplex RNA is preferred. The enzyme displays a profound base specificity, showing no activity on runs of C residues. RNase BN is strongly inhibited by the presence of a 3'-CCA sequence or a 3'-phosphoryl group. Digestion by RNase BN leads to 3-mers as the limit products, but the rate slows on molecules shorter than 10 nucleotides in length. Most interestingly, RNase BN acts as a distributive exoribonuclease on some substrates, releasing mononucleotides and a ladder of digestion products. However, RNase BN also cleaves endonucleolytically, releasing 3' fragments as short as 4 nucleotides. Although the presence of a 3'-phosphoryl group abolishes exoribonuclease action, it has no effect on the endoribonucleolytic cleavages. These data suggest that RNase BN may differ from other members of the RNase Z family, and they provide important information to be considered in identifying a physiological role for this enzyme.
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PMID:Catalytic properties of RNase BN/RNase Z from Escherichia coli: RNase BN is both an exo- and endoribonuclease. 1936 4

Here we report a unique processing pathway in Escherichia coli for tRNA(Leu5) in which the exoribonuclease polynucleotide phosphorylase (PNPase) removes the Rho-independent transcription terminator from the leuX transcript without requiring the RhlB RNA helicase. Our data demonstrate for the first time that PNPase can efficiently degrade an RNA substrate containing secondary structures in vivo. Furthermore, RNase P, an endoribonuclease that normally generates the mature 5'-ends of tRNAs, removes the leuX terminator inefficiently independent of PNPase activity. RNase P cleaves 4-7 nt downstream of the CCA determinant generating a substrate for RNase II, which removes an additional 3-4 nt. Subsequently, RNase T completes the 3' maturation process by removing the remaining 1-3 nt downstream of the CCA determinant. RNase E, G and Z are not involved in terminator removal. These results provide further evidence that the E. coli tRNA processing machinery is far more diverse than previously envisioned.
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PMID:Processing of the Escherichia coli leuX tRNA transcript, encoding tRNA(Leu5), requires either the 3'-->5' exoribonuclease polynucleotide phosphorylase or RNase P to remove the Rho-independent transcription terminator. 1990 95

RNase BN, the Escherichia coli homolog of RNase Z, was previously shown to act as both a distributive exoribonuclease and an endoribonuclease on model RNA substrates and to be inhibited by the presence of a 3'-terminal CCA sequence. Here, we examined the mode of action of RNase BN on bacteriophage and bacterial tRNA precursors, particularly in light of a recent report suggesting that RNase BN removes CCA sequences (Takaku, H., and Nashimoto, M. (2008) Genes Cells 13, 1087-1097). We show that purified RNase BN can process both CCA-less and CCA-containing tRNA precursors. On CCA-less precursors, RNase BN cleaved endonucleolytically after the discriminator nucleotide to allow subsequent CCA addition. On CCA-containing precursors, RNase BN acted as either an exoribonuclease or endoribonuclease depending on the nature of the added divalent cation. Addition of Co(2+) resulted in higher activity and predominantly exoribonucleolytic activity, whereas in the presence of Mg(2+), RNase BN was primarily an endoribonuclease. In no case was any evidence obtained for removal of the CCA sequence. Certain tRNA precursors were extremely poor substrates under any conditions tested. These findings provide important information on the ability of RNase BN to process tRNA precursors and help explain the known physiological properties of this enzyme. In addition, they call into question the removal of CCA sequences by RNase BN.
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PMID:Mode of action of RNase BN/RNase Z on tRNA precursors: RNase BN does not remove the CCA sequence from tRNA. 2048 3


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