Unstructured RNA Is a Substrate for tRNase Z

Shibata, Hirotaka S.; Minagawa, Asako; Takaku, Hiroaki; Takagi, Masamichi; Nashimoto, Masayuki

doi:10.1021/bi051972s

Cited by 19 publications

(23 citation statements)

References 25 publications

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“…7A that had 3 or 4 residues removed. These data confirm the conclusions of Shibata et al (20) that the enzyme can act as an endoribonuclease.…”

Section: Resultssupporting

confidence: 92%

“…These data were confirmed by experiments showing that RNase BN processed tRNA precursors lacking (15,16) or containing (8,19) the 3Ј-terminal CCA sequence in vivo. Other recent experiments showed that the E. coli enzyme could also act on some unstructured, synthetic RNAs, although most of these studies were carried out at 52°C (20). To examine in more detail the catalytic potential of RNase BN, we have carried out a systematic analysis of substrate specificity using a variety of defined, synthetic RNAs that enabled us to study separately various structural parameters.…”

Section: Resultsmentioning

confidence: 99%

“…However, because it is generally assumed that members of the RNase Z family are endoribonucleases (5), we wished to examine this point in more detail. For this purpose, we made use of a 24-nt synthetic RNA (usRNA1) previously studied by Shibata et al (20) with which it was concluded that the E. coli enzyme is an endoribonuclease. This RNA was labeled either at its 5Ј end with 32 P or at its 3Ј end with [ 32 P]pCp or [ 32 P]pC (following phosphatase treatment of the pCp-labeled molecule).…”

Section: Resultsmentioning

confidence: 99%

“…The 34-mer (G 5 A 29 ) or the 24-mer (usRNA1-5Ј-GAG UGA CUA CCU CCA AGG CCC UUU-3Ј) (20) 32 P]C) and 1.5 g of purified RNase BN. The reaction mixtures were incubated at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

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Catalytic Properties of RNase BN/RNase Z from Escherichia coli

Dutta

Deutscher

2009

Journal of Biological Chemistry

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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.Maturation of tRNA precursors requires the removal of 5Ј and 3Ј precursor-specific sequences to generate the mature, functional tRNA (1). In eukaryotes, archaea, and certain eubacteria, the 3Ј-processing step is carried out by an endoribonuclease termed RNase Z or 3Ј-tRNase (2-6). However, in some bacteria, such as Escherichia coli, removal of 3Ј extra residues is catalyzed by any of a number of exoribonucleases (7, 8). The major determinant for which mode of 3Ј-processing is utilized appears to be whether or not the universal 3Ј-terminal CCA sequence is encoded (2, 9). Thus, for those tRNA precursors in which the CCA sequence is absent, endonucleolytic cleavage by RNase Z right after the discriminator nucleotide generates a substrate for subsequent CCA addition by tRNA nucleotidyltransferase (1-3, 10). In view of this role for RNase Z in 3Ј-tRNA maturation, it is surprising that E. coli, an organism in which the CCA sequence is encoded in all tRNA genes (2), nevertheless contains an RNase Z homologue (11), because its action would appear not to be necessary. In fact, the physiological function of this enzyme in E. coli remains unclear, because mutants lacking this protein have no obvious growth phenotype (12). Hence, there is considerable interest in understanding the enzymatic capabilities of this enzyme.The E. coli RNase Z homologue initially was identified as a zinc phosphodiesterase (11) ...

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“…7A that had 3 or 4 residues removed. These data confirm the conclusions of Shibata et al (20) that the enzyme can act as an endoribonuclease.…”

Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Catalytic Properties of RNase BN/RNase Z from Escherichia coli

Dutta

Deutscher

2009

Journal of Biological Chemistry

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“…However, PhnP does exhibit regiospecific activity against 2Ј,3Ј-cyclic nucleotides. The E. coli tRNase ZipD is not active against 2Ј,3Ј-or 3Ј,5Ј-cyclic nucleotides (10) but will cleave short sequences of unstructured RNA (44). The noncatalytic Zn 2ϩ binding site of PhnP is one of the striking features of this structure, which, intriguingly, is shared by another "accessory" protein, PqqB, of the pyrroloquinoline quinone biosynthetic pathway.…”

Section: Discussionmentioning

confidence: 99%

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

Podzelinska

Wathier

et al. 2009

Journal of Biological Chemistry

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Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the ␤-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(pnitrophenyl)phosphate and 2,3-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn 2؉ and Ni 2؉ for catalysis, whereas Zn 2؉ afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 Å resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn 2؉ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn 2؉ binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.

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Theoretical Study of the RNA Hydrolysis Mechanism of the Dinuclear Zinc Enzyme RNase Z

Liao

Himo

et al. 2009

Eur J Inorg Chem

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RNase Z is a dinuclear zinc enzyme that catalyzes the removal of the tRNA 3Ј-end trailer. Density functional theory is used to investigate the phosphodiester hydrolysis mechanism of this enzyme with a model of the active site constructed on the basis of the crystal structure. The calculations imply that the reaction proceeds through two steps. The first step is a nucleophilic attack by a bridging hydroxide coupled with protonation of the leaving group by a Glu-His diad. Subsequently, a water molecule activated by the same Glu-His diad makes a reverse attack, regenerating the bridging hy-

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Unstructured RNA Is a Substrate for tRNase Z

Cited by 19 publications

References 25 publications

Catalytic Properties of RNase BN/RNase Z from Escherichia coli

Catalytic Properties of RNase BN/RNase Z from Escherichia coli

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

Theoretical Study of the RNA Hydrolysis Mechanism of the Dinuclear Zinc Enzyme RNase Z

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