ENZYMATIC FORMATION OF N-2,N-2-DIMETHYLGUANOSINE IN EUKARYOTIC TRANSFER-RNA - IMPORTANCE OF THE TRANSFER-RNA ARCHITECTURE

In eukaryotic tRNA, guanosine at position 26 in the junction between t he D-stem and the anticodon stem is mostly modified to N-2,N-2-dimethy lguanosine (m(2)(2)G(26)). Here we review the available information on the enzyme catalyzing the formation of this modified nucleoside, the SAM-dependent tRNA(m(2)(2)G(26))-methyltransferase, and our attemps to identify the parameters in tRNA needed for efficient enzymatic dimeth ylation of guanosine-26, The required identity elements in yeast tRNA for dimethylation under in vitro conditions by the yeast tRNA(m(2)(2)G (26))-methyltransferase (the TRM1 gene product) are comprised of two G -C base pairs at positions G(10)-C-25 and C-11-G(24) in the D-stem tog ether with a variable loop of at least five nucleotides. These positiv e determinants do not seem to act via base specific interactions with the methyltransferase; they instead ensure that G(26) is presented to the enzyme in a favourable orientation, within the central 3D-core of the tRNA molecule. The anticodon stem and loop is not involved in such an interaction with the enzyme. n a heterologous in vivo system, cons isting of yeast tRNAs microinjected into Xenopus laevis oocytes, the r equirements for modification of G(26) are less stringent than in the y east homologous in vitro system. Indeed, G(26) in several microinjecte d tRNAs becomes monomethylated, while in yeast extracts it stays unmet hylated, even after extensive incubation. Thus either the X laevis tRN A(m(2)(2)G(26))-methyltransferase has a more relaxed specificity than its yeast homolog, or there exist two distinct G(26)-methylating activ ities, one for G(26)-monomethylation, and one for dimethylation of G(2 6). Our results stress the importance of the local 3D-architecture of the tRNA substrate for efficient enzymatic formation of m(2)(2)G(26).