Structural features of tRNALys favored by anticodon nuclease as inferred from reactivities of anticodon stem and loop substrate analogs

Yue Jiang, Shani Blanga, Michal Amitsur, Roberto Meidler, Eli Krivosheyev, Mallikarjun Sundaram, Ashok C. Bajji, Darrell R. Davis, Gabriel Kaufmann

Research output: Contribution to journalArticlepeer-review

Abstract

The bacterial tRNALys-specific PrrC-anticodon nuclease efficiently cleaved an anticodon stem-loop (ASL) oligoribonucleotide containing the natural modified bases, suggesting this region harbors the specificity determinants. Assays of ASL analogs indicated that the 6-threonylcarbamoyl adenosine modification (t6A37) enhances the reactivity. The side chain of the modified wobble base 5-methylaminomethyl-2-thiouridine (mnm5s2U34) has a weaker positive effect depending on the context of other modifications. The s2U34 modification apparently has none and the pseudouridine (ψ39) was inhibitory in most modification contexts. GC-rich but not IC-rich stems abolished the activity. Correlating the reported structural effects of the base modifications with their effects on anticodon nuclease activity suggests preference for substrates where the anticodon nucleotides assume a stacked A-RNA conformation and base pairing interactions in the stem are destabilized. Moreover, the proposal that PrrC residue Asp287 contacts mnm5s2U34 was reinforced by the observations that the mammalian tRNALys-3 wobble base 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U) is inhibitory and that the D287H mutant favors tRNALys-3 over Escherichia coli tRNALYs. The detection of this mutation and ability of PrrC to cleave the isolated ASL suggest that anticodon nuclease may be used to cleave tRNALys-3 primer molecules annealed to the genomic RNA template of the human immunodeficiency virus.

Original languageEnglish
Pages (from-to)3836-3841
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number6
DOIs
StatePublished - 8 Feb 2002

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