title: Enzymatic formation of N2,N2-dimethylguanosine in eukaryotic tRNA: importance of the tRNA architecture.
abstract: In eukaryotic tRNA, guanosine at position 26 in the junction between the D-stem and the anticodon stem is mostly modified to N2,N2-dimethylguanosine (m2(2)G26). Here we review the available information on the enzyme catalyzing the formation of this modified nucleoside, the SAM-dependent tRNA (m2(2)G26)-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(m2(2)G26)-methyltransferase (the TRM1 gene product) are comprised of two G-C base pairs at positions G10-C25 and C11-G24 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 G26 is presented to the enzyme in a favorable 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. In a heterologous in vivo system, consisting of yeast tRNAs microinjected into Xenopus laevis oocytes, the requirements for modification of G26 are less stringent than in the yeast homologous in vitro system. Indeed, G26 in several microinjected tRNAs becomes monomethylated, while in yeast extracts it stays unmethylated, even after extensive incubation. Thus either the X laevis tRNA(m2(2)G26)-methyltransferase has a more relaxed specificity than its yeast homolog, or there exist two distinct G26-methylating activities, one for G26-monomethylation, and one for dimethylation of G26.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Microbiology, University of Umeå, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Edqvist
authors.forename: K B
authors.initials: KB
authors.lastname: Stråby
authors.forename: H
authors.initials: H
authors.lastname: Grosjean
mesh_terms: D006151:Guanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D008745:Methylation; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics; D012359:tRNA Methyltransferases / Q000378:metabolism*
chemical_list: D012331:RNA, Fungal; D006151:Guanosine; C008940:N(2),N(2)-dimethylguanosine; D012436:S-Adenosylmethionine; D012343:RNA, Transfer; C059863:N(2),N(2)-dimethylguanosine-specific tRNA methyltransferase; D012359:tRNA Methyltransferases