Structure–activity relationships in <i>Kluyveromyces lactis</i> γ-toxin, a eukaryal tRNA anticodon nuclease

Keppetipola, Niroshika; Jain, Ruchi; Meineke, Birthe; Diver, Melinda M.; Shuman, Stewart

doi:10.1261/rna.1637809

Cited by 22 publications

(25 citation statements)

References 35 publications

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“…Like other metal-independent ribonucleases, Cas6 likely cleaves RNAs via a transesterification reaction employing general acid-base chemistry (Steyaert 1997;Raines 1998;Xue et al 2006;Yajima et al 2006;Keppetipola et al 2009). The available evidence suggests that Cas6 shares similar active-site architecture and likely reaction characteristics with the archaeal tRNA splicing endonuclease (Xue et al 2006;.…”

Section: Suggested Cas6 Crispr Rna Cleavage Mechanismmentioning

confidence: 99%

“…The Cas6 protein contains a characteristic glycine-rich, GhGxxxxGhG motif (where h indicates a hydrophobic amino acid and xxxx includes at least one lysine or arginine) present at the C terminus (Haft et al 2005). Cas6 cleavage activity is independent of divalent metal ions and generates products with 59-hydroxyl and likely 29-39 cyclic phosphate ends (Carte et al 2008), typical of metal-independent ribonucleases (Steyaert 1997;Raines 1998;Xue et al 2006;Yajima et al 2006;Keppetipola et al 2009). Three conserved residues (Tyr, His, and Lys) that are separated from one another in the primary sequences of Cas6 proteins from diverse organisms were found to cluster in the crystal structure of Cas6 from P. furiosus (Carte et al 2008).…”

Section: Introductionmentioning

confidence: 99%

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Binding and cleavage of CRISPR RNA by Cas6

Carte¹,

Pfister²,

Compton³

et al. 2010

RNA

146

158

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The CRISPR-Cas system provides many prokaryotes with acquired resistance to viruses and other mobile genetic elements. The core components of this defense system are small, host-encoded prokaryotic silencing (psi)RNAs and Cas (CRISPR-associated) proteins. Invader-derived sequences within the psiRNAs guide Cas effector proteins to recognize and silence invader nucleic acids. Critical for CRISPR-Cas defense is processing of the psiRNAs from the primary transcripts of the host CRISPR (clustered regularly interspaced short palindromic repeat) locus. Cas6, a previously identified endoribonuclease present in a wide range of prokaryotes with the CRISPR-Cas system, binds and cleaves within the repeat sequences that separate the individual invader targeting elements in the CRISPR locus transcript. In the present study, we investigated several key aspects of the mechanism of function of Cas6 in psiRNA biogenesis. RNA footprinting reveals that Pyrococcus furiosus Cas6 binds to a 7-nt (nucleotide) sequence near the 59 end of the CRISPR RNA repeat sequence, 14 nt upstream of the Cas6 cleavage site. In addition, analysis of the cleavage activity of P. furiosus Cas6 proteins with mutations at conserved residues suggests that a triad comprised of Tyr31, His46, and Lys52 plays a critical role in catalysis, consistent with a possible general acid-base RNA cleavage mechanism for Cas6. Finally, we show that P. furiosus Cas6 remains stably associated with its cleavage products, suggesting additional roles for Cas6 in psiRNA biogenesis.

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Section: Suggested Cas6 Crispr Rna Cleavage Mechanismmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Binding and cleavage of CRISPR RNA by Cas6

Carte¹,

Pfister²,

Compton³

et al. 2010

RNA

146

158

View full text Add to dashboard Cite

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“…Biochemical characterization of recombinant g-toxin highlighted its RNase A-like chemical mechanism and a putative role of the essential His209 residue (the only histidine present in g-toxin) as a general acid or general base in catalysis of transesterification. Incision of the 17-mer stem-loop by g-toxin was abolished by ribose 29-H or 29-F modifications of the wobble uridine (Keppetipola et al 2009), as expected for a site-specific transesterifying endonuclease that recognizes only one scissile phosphodiester in the tRNA substrate.…”

Section: Introductionmentioning

confidence: 52%

“…1; Lu et al 2008). The unmodified 17-mer RNA (R17), when 59 32 P-labeled, is incised by g-toxin at the correct phosphodiester 39 of the first uridine of the UUC anticodon to generate a single 32 Plabeled 8-mer product (Lu et al 2008;Keppetipola et al 2009). The minimized R17 substrate has been extremely useful in dissecting the influence of nucleobase sequence on substrate utilization.…”

Section: Introductionmentioning

confidence: 99%

“…S1). We proceeded to determine their relevant properties by testing the effects of conservative substitutions for each of the essential amino acids (Keppetipola et al 2009). Biochemical characterization of recombinant g-toxin highlighted its RNase A-like chemical mechanism and a putative role of the essential His209 residue (the only histidine present in g-toxin) as a general acid or general base in catalysis of transesterification.…”

Section: Introductionmentioning

confidence: 99%

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Substrate specificity and mutational analysis of Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease

Jain

Poulos²,

Gros³

et al. 2011

RNA

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tRNA anticodon damage inflicted by the Kluyveromyces lactis g-toxin underlies an RNA-based innate immune system that distinguishes self from nonself species. g-toxin arrests the growth of Saccharomyces cerevisiae by incising a single phosphodiester 39 of the wobble base of tRNA Glu(UUC) to generate a break with 29,39-cyclic phosphate and 59-OH ends. Recombinant g-toxin cleaves oligonucleotide substrates in vitro that mimic the anticodon stem-loop of tRNA Glu . A single 29-deoxy sugar substitution at the wobble nucleoside abolishes anticodon nuclease activity. To gain further insights to g-toxin's substrate specificity, we tested deoxynucleoside effects at positions other than the site of transesterification. The results attest to a stringent requirement for a ribonucleoside at the uridine 59 of the wobble base. In contrast, every other nonwobble ribonucleoside in the anticodon loop can be replaced by a deoxy without significantly affecting g-toxin's cleavage activity. Whereas either the 59 half or the 39 half of the anticodon stem can be replaced en bloc with DNA without a major effect, simultaneously replacing both strands with DNA interfered strongly, signifying that g-toxin requires an A-form helical conformation of the anticodon stem. We purified g-toxin mutants identified previously as nontoxic in vivo and gauged their anticodon nuclease activities in vitro. The results highlight Glu9 and Arg151 as candidate catalytic residues, along with His209 implicated previously. By analogy to other endoribonucleases, we speculate that g-toxin drives transesterification by general acid-base catalysis (via His209 and Glu9) and transition-state stabilization (via Arg151).

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RNA toxins: mediators of stress adaptation and pathogen defense

et al. 2011

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RNA toxins are a group of enzymes primarily synthesized by bacteria, fungi, and plants that either cleave or depurinate RNA molecules. These proteins may be divided according to their RNA substrates: ribotoxins are nucleases that cleave ribosomal RNA (rRNA), ribosome inactivating proteins are glycosidases that remove a base from rRNA, messenger RNA (mRNA) interferases are nucleases that cleave mRNAs, and anticodon nucleases cleave transfer RNAs (tRNAs). These modifications to the RNAs may substantially alter gene expression and translation rates. Given that some of these enzymes cause cell death, it has been suggested that they function mainly in defense, either to kill competing cells or to elicit suicide and thereby limit pathogen spread from infected cells. Although good correlations have been drawn between their enzymatic functions and toxicity, recent work has shown that some RNA toxins cause apoptosis in the absence of damage to RNA and that defense against pathogens can be achieved without host cell death. Moreover, a decrease in cellular translation rate, insufficient to cause cell death, allows some organisms to adapt to stress and environmental change. Although ascribing effects observed in vitro to the roles of these toxins in nature has been challenging, recent results have expanded our understanding of their modes of action, and emphasized the importance of these toxins in development, adaptation to stress and defense against pathogens.

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Structure–activity relationships in Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease

Cited by 22 publications

References 35 publications

Binding and cleavage of CRISPR RNA by Cas6

Binding and cleavage of CRISPR RNA by Cas6

Substrate specificity and mutational analysis of Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease

RNA toxins: mediators of stress adaptation and pathogen defense

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