Nucleic Acids Research, Vol 27, Issue 5 1308-1315, Copyright © 1999 by Oxford University Press
F Constantinesco, Y Motorin and H Grosjean
The modification patterns of in vitro transcripts of two yeast
Saccharomyces cerevisiae tRNAs (tRNAPheand tRNAAsp) and one archaeal
Haloferax volcanii tRNA (tRNAIle) were investigated in the cell-free
extract of Pyrococcus furiosus supplemented with S -adenosyl-l- methionine
(AdoMet). The results indicate that the enzymatic formation of 11 distinct
modified nucleotides corresponding to 12 enzymatic activities can be
detected in vitro. They correspond to the formation of pseudouridines (Psi)
at positions 39 and 55, 2' -O- ribose methylations at positions 6 (Am) and
56 (Cm), base methylations at positions 10 (m2G), 26 (m22G), 37 (m1G), 49
(m5C), 54 (m5U) and 58 (m1A) and both the deamination and methylation of
adenosine into m1I at position 57. Most of the detected modified
nucleotides are common modifications found in other phylogenetic groups,
while Am6, Cm56and m1I57are specific modifications found exclusively in
Archaea. It is also shown that the enzymatic formation of m5C49, m5U54,
Psi55and m1I57does not depend on the three-dimensional architecture of the
tRNA substrate, since these modi-fications also occur in fragmented tRNAs
as substrate.
ARTICLES
Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro
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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