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

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.