Characterisation and enzymatic properties of tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) from Pyrococcus furiosus.
The structural gene TRM1 encoding tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and expressed in Escherichia coli. The corresponding recombinant enzyme (pfTrm1p) with a His6-tag at the N terminus was purified to homogeneity in three steps. The enzyme has a native molecular mass of 49 kDa (as determined by gel filtration) and is very stable to heat denaturation (t1/2at 95 degrees C is two hours). pfTrm1p is a monomer and forms a one to one complex with T7 transcripts of yeast tRNA(Phe). It methylates a single guanine residue at position 26 using S -adenosyl- l -methionine as donor of the methyl groups. Depending on the incubation temperature, the type of tRNA transcript and the ratio of enzyme to tRNA, m(2)G26 or m(2)2G26 was the main product. The addition of the second methyl group to N (2)guanine 26 takes place in vitro through a monomethylated intermediate, and the enzyme dissociates from its tRNA substrate between the two consecutive methylation reactions. Identity elements in tRNA for mono- and dimethylation reactions by the recombinant pfTrm1p were identified using in vitro T7 transcripts of 33 variants of tRNA(Asp)and tRNA(Phe)from yeast. The efficient dimethylation of G26 requires the presence of base-pairs C11.G24 and G10.C25 and a variable loop of five bases within a correct 3D-core of the tRNA molecule. These identity elements probably ensure the correct presentation of monomethylated m(2)G26 to the enzyme for the attachment of the second methyl group. In contrast, the structural requirements for monomethylation of the same guanine 26 are much more relaxed and tolerate variations in the base-pairs of the D-stem, in the size of the variable loop or distortions of the 3D-architecture of the tRNA molecule.
Constantinesco
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F
Laboratoire d'Enzymologie et Biochimie Structurales, C.N.R.S., 1 av. de la Terrasse, Gif-sur-Yvette, F-91198, France.
Motorin
Y
Y
Grosjean
H
H
D003001:Cloning, Molecular; D004795:Enzyme Stability; D006147:Guanine / Q000378:metabolism; D006361:Heating; D006639:Histidine; D007700:Kinetics; D009690:Nucleic Acid Conformation; D020027:Pyrococcus furiosus / Q000201:enzymology*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
D012360:RNA, Transfer, Phe; D011993:Recombinant Fusion Proteins; D006639:Histidine; D006147:Guanine; C089041:N(2),N(2)-dimethylguanosine-26-methyltransferase; D012359:tRNA Methyltransferases