Nucleic Acids Research, Vol 26, Issue 22 5102-5108, Copyright © 1998 by Oxford University Press
J Liu, J Liu and KB Straby
Guanosine at position 26 in eukaryotic tRNAs is usually modified to N2 , N2
-dimethylguanosine (m22G26). In Saccharomyces cerevisiae , this reaction is
catalysed by the TRM1 encoded tRNA (m22G26)dimethyltransferase. As a
prerequisite for future studies, the yeast TRM1 gene was expressed in
Escherichia coli and the His-tagged Trm1 protein (rTrm1p) was extensively
purified. rTrm1p catalysed both the mono- and dimethylation of G26 in vivo
in Escherichia coli tRNA and in vitro in yeast trm1 mutant tRNA. The TRM1
gene from two independent wild-type yeast strains differed at 14 base
positions causing two amino acid exchanges . Exchange of the original
Ser467 for Leu caused a complete loss of enzyme activity in vitro against
trm1 yeast tRNA. Comparatively short N- or C-terminal deletions from the
570 amino acid long Trm1 polypeptide decreased or eliminated the enzyme
activity, as did some point mutations within these regions. This indicated
that the protein is not a two domain peptide with the enzyme activity
localised to one of the domains, but rather that both ends of the
polypeptide seem to interact to influence the conformation of those parts
that make up the RNA-binding site and/or the active site of the enzyme.
ARTICLES
Point and deletion mutations eliminate one or both methyl group transfers catalysed by the yeast TRM1 encoded tRNA (m22G26)dimethyltransferase
Department of Microbiology, Umea University, S-901 87 Umea, Sweden and Shanghai Research Centre of Life Sciences, Academia Sinica, 200031 Shanghai, China.
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