Detection of anticodon nuclease residues involved in tRNALys cleavage specificity

Meidler, Roberto; Morad, Ilan; Amitsur, Michal; Inokuchi, Hachiro; Kaufmann, Gabriel

doi:10.1006/jmbi.1999.2634

Cited by 24 publications

(54 citation statements)

References 33 publications

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“…Overexpressing PrrCs with inactivating mutations in the NTPase active site ameliorated the toxicity of wild-type EcoPrrC; these dominant negative effects were not observed with PrrCs containing inactivating mutations in the nuclease domain. Our findings, building on the elegant studies of EcoPrrC by Gabi Kaufmann and colleagues (Meidler et al 1999;Jiang et al 2001Jiang et al , 2002Amitsur et al 2003;Blanga-Kanfi et al 2006), support a model in which EcoPrrC toxicity is contingent on head-to-tail dimerization of the ABC-like NTPase domains and the consequent formation of two composite NTP phosphohydrolase active sites, which in turn activates the nuclease domains in cis, only one of which needs be functional.…”

Section: Introductionsupporting

confidence: 72%

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

Meineke

Shuman²

2011

RNA

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Breakage of tRNA Lys(UUU) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNA Lys(UUU) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 59-kinase and ligase functions.

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Section: Introductionsupporting

confidence: 72%

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

Meineke

Shuman²

2011

RNA

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“…In this context, the anticodon nuclease PrrC is another interesting cytotoxic tRNase, although it is not a physiological toxin. PrrC is produced by a clinical E. coli strain in response to phage T4 infection and specifically cleaves intracellular tRNA Lys between positions 33 and 34 to interfere with propagation of the infected phage (32)(33)(34). Despite apparently similar activities toward tRNAs, again significant homology has not been found between PrrC and colicin E5 or D.…”

Section: Discussionmentioning

confidence: 99%

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

Tomita

Ogawa

Uozumi

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

158

143

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Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAs Arg including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2 ,3 -cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAs Arg , the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNA Arg -cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAs Arg , which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.C olicins are plasmid-encoded proteins that are toxic to Escherichia coli cells that do not have the same plasmid or a cognate Col plasmid (1-5). Most colicins are produced in response to SOS-inducing signals and are secreted into the medium. After binding to cell-surface receptors on sensitive cells, they are translocated across the membrane and then exert their final cytotoxic activities, which are attributable to their C-terminal domains. Two major modes of toxicity are well known; colicins A, B, E1, Ia, Ib, K, and N are ion-channel formers attacking the cytoplasmic membrane, and colicins E2 to E9 are nucleases. In the latter group, E2, E7, E8, and E9 are DNases, and E3 is a special kind of RNase that cleaves 16S rRNA within ribosomes.Colicins E4 to E6 quickly stop amino acid incorporation in treated cells (6), suggesting impairment of protein synthesis analogous in the established case of E3, which specifically cleaves 16S-RNA at the 49th bond from the 3Ј end leading to inactivation of ribosomes (7-9). This is also the case with colicin D, which Timmis and Hedges have characterized as to the physiological response of treated cells (10, 11), although the actual molecular basis of the cytotoxic effect of colicin D, as well as those of E4 to E6, remained to be elucidated. Colicins E4 and E6 proved to be E3-homologs and showed comparable activity toward ribosomes (ref. 12; GenBank accession number X63621; Y. Gunji, M. Ohno, T.O., H.M., and T.U., unpublished data), but colicin E5 exhibits no similarity to E3 in the C-terminal active domain. We recently showed that colicin E5 comprises a third category of nuclease-type colicins, which does not attack ribosomes but specific tRNAs (13). E5 is a novel RNase that cleaves the anticodons...

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“…Mutating one of them, EcoPrrC's Asp 287 impairs the reactivity of the natural substrate and enhances that of analogs with a hypomodified or heterologous wobble base. These compensations hint that Asp 287 interacts with the wobble base modifying side chain (Meidler et al, 1999;Jiang et al, 2001;Jiang et al, 2002). When PrrC is expressed by itself it exhibits overt (core) ACNase activity.…”

Section: Prrc's Functional Organizationmentioning

confidence: 99%

“…PrrC's dissociation from the latent holoenzyme may be accidental or due to EcoprrI's disruption in response to DNA damage (Restriction Alleviation, RA) (Makovets et al, 2004) (see also section 3.6). Free PrrC's cytotoxicity has been indicated by the coincident inactivation of prrC and linked hsd genes, by the self-limiting expression of free PrrC (Meidler et al, 1999;Blanga-Kanfi et al, 2006) and the rapid in vivo inactivation of the core ACNase (Amitsur et al, 2003). The ACNase enhancing effects of dTTP's accretion during phage T4 infection (Klaiman & Kaufmann, 2011) and in vitro stabilization of the core ACNase by dTTP (Amitsur et al, 2003) suggest that the in vivo instability of the core ACNase owes to the relatively low dTTP level in the uninfected cell.…”

Section: Players In Prrc's Silencing and Activationmentioning

confidence: 99%

“…However, identifying RloC's natural substrate must await physiological studies. This reservation is based on the experience gained with PrrC, the over-expression of which results in cleavages of secondary substrates that overwhelm the natural (Meidler et al, 1999). A more striking difference between RloC and PrrC is the ability of the former to cleave its tRNA substrates successively, first 3' and then 5' to the wobble position (Davidov & Kaufmann, 2008).…”

Section: Rloc Wobble-nucleotide-excising Activitymentioning

confidence: 99%

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RloC: A Translation-Disabling tRNase Implicated in Phage Exclusion During Recovery from DNA Damage

Kaufmann¹,

Davidov²,

Steinfels-Kohn³

et al. 2011

DNA Repair

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Detection of anticodon nuclease residues involved in tRNALys cleavage specificity

Cited by 24 publications

References 33 publications

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

RloC: A Translation-Disabling tRNase Implicated in Phage Exclusion During Recovery from DNA Damage

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