Structural features of tRNALys favored by anticodon nuclease as inferred from reactivities of anticodon stem and loop substrate analogs.
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.
Jiang
Yue
Y
Department of Biochemistry, Tel Aviv University, Ramat, Aviv 69978, Israel.
Blanga
Shani
S
Amitsur
Michal
M
Meidler
Roberto
R
Krivosheyev
Eli
E
Sundaram
Mallikarjun
M
Bajji
Ashok C
AC
Davis
Darrell R
DR
Kaufmann
Gabriel
G
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
D012357:RNA, Transfer, Lys; D012260:Ribonucleases; C049345:anticodon nuclease