Substrate specificity and mutational analysis of Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease Journal Article
| Authors: | Jain, R.; Poulos, M. G. ; Gros, J.; Chakravarty, A. K.; Shuman, S. |
| Article Title: | Substrate specificity and mutational analysis of Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease |
| Abstract: | tRNA anticodon damage inflicted by the Kluyveromyces lactis γ-toxin underlies an RNA-based innate immune system that distinguishes self from nonself species. γ-toxin arrests the growth of Saccharomyces cerevisiae by incising a single phosphodiester 3′ of the wobble base of tRNA Glu(UUC) to generate a break with 2′,3′-cyclic phosphate and 5′-OH ends. Recombinant γ-toxin cleaves oligonucleotide substrates in vitro that mimic the anticodon stem-loop of tRNAGlu. A single 2′-deoxy sugar substitution at the wobble nucleoside abolishes anticodon nuclease activity. To gain further insights to γ-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 5′ of the wobble base. In contrast, every other nonwobble ribonucleoside in the anticodon loop can be replaced by a deoxy without significantly affecting γ-toxin's cleavage activity. Whereas either the 5′ half or the 3′ 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 γ-toxin requires an A-form helical conformation of the anticodon stem. We purified γ-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 γ-toxin drives transesterification by general acid-base catalysis (via His209 and Glu9) and transition-state stabilization (via Arg151). Published by Cold Spring Harbor Laboratory Press. Copyright © 2011 RNA Society. |
| Keywords: | unclassified drug; nonhuman; enzyme activity; tyrosine; nuclease; catalysis; transfer rna; enzyme specificity; glutamic acid; arginine; gamma toxin; histidine; mycotoxin; anticodon; transesterification; kluyveromyces lactis; enzyme stability; dna strand; ribotoxin; trna anticodon stem-loop; ribonucleoside; fungus mutant |
| Journal Title: | RNA |
| Volume: | 17 |
| Issue: | 7 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2011-07-01 |
| Start Page: | 1336 |
| End Page: | 1343 |
| Language: | English |
| DOI: | 10.1261/rna.2722711 |
| PROVIDER: | scopus |
| PMCID: | PMC3138569 |
| PUBMED: | 21610213 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 17 August 2011" - "CODEN: RNARF" - "Source: Scopus" |
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