Methylation of the ribosyl moiety at position 34 of selenocysteine tRNA([Ser]Sec) is governed by both primary and tertiary structure

The selenocysteine (Sec) tRNA([Ser]Sec) population in higher vertebrates co nsists of two major isoacceptors that differ from each other by a single nu cleoside modification in the wobble position of the anticodon (position 34) , One isoacceptor contains 5-methylcarboxymethyluridine (mcmU) in this posi tion, whereas the other contains 5-methylcarboxymethyluridine-2'-O-methylri bose (mcmUm), The other modifications in these tRNAs are N-6-isopentenylade nosine (i(6)A), pseudouridine (psi), and 1-methyladenosine (m(1)A) at posit ions 37, 55, and 58, respectively, As methylation of the ribose at position 34 is influenced by the intracellular selenium status and the presence of this methyl group dramatically alters tertiary structure, we investigated t he effect of the modifications at other positions as well as tertiary struc ture on its formation, Mutations were introduced within a synthetic gene en coded in an expression vector, transcripts generated and microinjected into Xenopus oocytes, and the resulting tRNA products analyzed for the presence of modified bases, The results suggest that efficient methylation of mcmU to yield mcmUm requires the prior formation of each modified base and an in tact tertiary structure, whereas formation of modified bases at other posit ions, including mcmU, is not as stringently connected to precise primary an d tertiary structure, These results, along with the observations that methy lation of mcmU is enhanced in the presence of selenium and that this methyl group affects tertiary structure, further suggest that the mcmUm isoaccept or must have a role in selenoprotein synthesis different from that of the m cmU isoacceptor.