Transfer RNA modification enzymes from Pyrococcus furiosus:: detection of the enzymatic activities in vitro

The modification patterns of in vitro transcripts of two yeast Saccharomyces cerevisiae tRNAs (tRNA(Phe) and tRNA(Asp)) and one archaeal Haloferax volcanii tRNA (tRNA(Ile)) were investigated in the cell-free extract of Pyrococcus furiosus supplemented with Sadenosyl-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 (m(2)G), 26 (m(2)(2)G), 37 (m(1)G), 49 (m(5)C), 54 (m(5)U) and 58 (m(1)A) and both the deamination and methylation of adenosine into m(1)l at position 57, Most of the detected modified nucleotides are common modifications found in other phylogenetic groups, while Am-6, Cm-56 and m(1)l(57) are specific modifications found exclusively in Archaea. It is also shown that the enzymatic formation of m(5)C(49), m(5)U(54), Psi(55) and m(1)l(57) does not depend on the three-dimensional architecture of the tRNA substrate, since these modifications also occur in fragmented tRNAs as substrate.