Nucleic Acids Research, Vol 26, Issue 16 3753-3761, Copyright © 1998 by Oxford University Press
F Constantinesco, N Benachenhou, Y Motorin and H Grosjean
The structural gene pfTRM1 (GenBank accession no. AF051912), encoding
tRNA(guanine-26, N 2- N 2) methyltransferase (EC 2.1.1.32) of the strictly
anaerobic hyperthermophilic archaeon Pyrococcus furiosus, has been
identified by sequence similarity to the TRM1 gene of Saccharomyces
cerevisiae (YDR120c). The pfTRM1 gene in a 3.0 kb restriction DNA fragment
of P.furiosus genomic DNA has been cloned by library screening using a PCR
probe to the 5'-part of the corresponding ORF. Sequence analysis revealed
an entire ORF of 1143 bp encoding a polypeptide of 381 residues (calculated
molecular mass 43.3 kDa). The deduced amino acid sequence of this newly
identified gene shares significant similarity with the TRM1- like genes of
three other archaea (Methanococcus jannaschii, Methanobacterium
thermoautotrophicum and Archaeoglobus fulgidus), one eukaryon
(Caenorhabditis elegans) and one hyperthermophilic eubacterium (Aquifex
aeolicus). Two short consensus motifs for S-adenosyl-l-methionine binding
are detected in the sequence of pfTrm1p. Cloning of the P.furiosus TRM1
gene in an Escherichia coli expression vector allowed expression of the
recombinant protein (pfTrm1p) with an apparent molecular mass of 42 kDa. A
protein extract from the transformed E.coli cells shows enzymatic activity
for the quantitative formation of N 2, N 2-dimethylguanosine at position 26
in a transcript of yeast tRNAPhe used as substrate. The recombinant enzyme
was also shown to modify bulk E.coli tRNAs in vivo.
ARTICLES
The tRNA(guanine-26,N2-N2) methyltransferase (Trm1) from the hyperthermophilic archaeon Pyrococcus furiosus: cloning, sequencing of the gene and its expression in Escherichia coli
Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique,1 Avenue de la Terrasse, Batiment 34, F-91198 Gif-sur-Yvette, France.
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