2'-O-methylation and inosine formation in the wobble position of anticodon-substituted tRNA-Phe in a homologous yeast in vitro system.
Four variants of yeast tRNA-Phe in which the anticodon and 3'-adjacent nucleotide (GmAAY) have been replaced by synthetic tetranucleotides NAAG (where N is each of the four canonical nucleosides G, C, U or A) are substrates for a yeast tRNA modification enzyme which catalyses the S-adenosyl-L-methionine dependent formations of Gm-34, Cm-34, Um-34, Am-34 and Im-34 (where Nm represents a 2'-O-methylnucleoside and I inosine). The kinetics of these nucleosides-34 2'-O-methylations reveal that yeast tRNA-Phe with G-34 (the natural substrate) is less efficiently modified than variants of the same tRNA containing U-34 and C-34. The formation of Am-34 in the tRNA containing A-34 was found to be particularly inefficient. However, in this tRNA, we observed the formation of I-34 followed by a 2'-O-methylation (giving rise to Im-34). In the yeast in vitro system described here, inosine formation is not dependent on the addition of any cofactor including hypoxanthine; the mechanism of inosine formation in yeast tRNA might therefore be distinct from that found in higher eukaryotes.
Droogmans
L
L
Laboratoire de Chimie Biologique, Université Libre de Bruxelles, Rhode-St-Genèse, Belgium.
Grosjean
H
H
D000926:Anticodon / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D007288:Inosine / Q000096:biosynthesis*; D008745:Methylation; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D000926:Anticodon; D012360:RNA, Transfer, Phe; D007288:Inosine