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 the D-stem and the anticodon stem is mostly modified to N-2,N-2-dimethylguanosine (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 dimethylation 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 together with a variable loop of at least five nucleotides. These positive 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, consisting of yeast tRNAs microinjected into Xenopus laevis oocytes, the requirements for modification of G(26) are less stringent than in the yeast homologous in vitro system. Indeed, G(26) in several microinjected tRNAs becomes monomethylated, while in yeast extracts it stays unmethylated, even after extensive incubation. Thus either the X laevis tRNA(m(2)(2)G(26))-methyltransferase has a more relaxed specificity than its yeast homolog, or there exist two distinct G(26)-methylating activities, one for G(26)-monomethylation, and one for dimethylation of G(26). Our results stress the importance of the local 3D-architecture of the tRNA substrate for efficient enzymatic formation of m(2)(2)G(26).