title: Structural features of tRNALys favored by anticodon nuclease as inferred from reactivities of anticodon stem and loop substrate analogs.
abstract: The bacterial tRNA(Lys)-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 (t(6)A37) enhances the reactivity. The side chain of the modified wobble base 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) has a weaker positive effect depending on the context of other modifications. The s(2)U34 modification apparently has none and the pseudouridine (psi39) 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 Asp(287) contacts mnm(5)s(2)U34 was reinforced by the observations that the mammalian tRNA(Lys-3) wobble base 5-methoxycarbonyl methyl-2-thiouridine (mcm(5)s(2)U) is inhibitory and that the D287H mutant favors tRNA(Lys-3) over Escherichia coli tRNA(Lys). The detection of this mutation and ability of PrrC to cleave the isolated ASL suggest that anticodon nuclease may be used to cleave tRNA(Lys-3) primer molecules annealed to the genomic RNA template of the human immunodeficiency virus.
authors.affiliation: Department of Biochemistry, Tel Aviv University, Ramat, Aviv 69978, Israel.
authors.forename: Yue
authors.initials: Y
authors.lastname: Jiang
authors.forename: Shani
authors.initials: S
authors.lastname: Blanga
authors.forename: Michal
authors.initials: M
authors.lastname: Amitsur
authors.forename: Roberto
authors.initials: R
authors.lastname: Meidler
authors.forename: Eli
authors.initials: E
authors.lastname: Krivosheyev
authors.forename: Mallikarjun
authors.initials: M
authors.lastname: Sundaram
authors.forename: Ashok C
authors.initials: AC
authors.lastname: Bajji
authors.forename: Darrell R
authors.initials: DR
authors.lastname: Davis
authors.forename: Gabriel
authors.initials: G
authors.lastname: Kaufmann
mesh_terms: D007700:Kinetics; D009154:Mutation; D009690:Nucleic Acid Conformation; D012357:RNA, Transfer, Lys / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012357:RNA, Transfer, Lys; D012260:Ribonucleases; C049345:anticodon nuclease