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

The modification patterns of in vitro transcripts of two yeast Saccharomyce s cerevisiae tRNAs (tRNA(Phe) and tRNA(Asp)) and one archaeal Haloferax vol canii tRNA (tRNA(Ile)) were investigated in the cell-free extract of Pyroco ccus furiosus supplemented with Sadenosyl-L-methionine (AdoMet). The result s indicate that the enzymatic formation of 11 distinct modified nucleotides corresponding to 12 enzymatic activities can be detected in vitro. They co rrespond 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 m odifications 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 s hown 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 sub strate, since these modifications also occur in fragmented tRNAs as substra te.