Solution Structure of Selenocysteine-inserting tRNA Sec from Escherichia coli: Comparison with Canonical tRNA Ser

Abstract

Selenocysteine-inserting tRNs (or tRNA Sec) are structurally untypical tRNAs that are charged by seryl-tRNA synthetase before being recognized by the selenocysteine synthase that converts serine into selenocysteine. tRNA Sec from Escherichia coli contains 95 nucleotides and is the longest known to date, in contrast to canonical tRNA Ser, 88 nucleotides-long. We have studied its solution conformation by chemical and enzymatic probing. Global structural features were obtained by cobra venom and S 1 nuclease mapping, as well as by probing with Pb 2+. Accessibilities of phosphate groups were measured by ethylnitrosourea probing. Information about positions in bases involved in Watson-Crick pairing, in stacking or in tertiary interactions were obtained by chemical probing with dimethylsulfate, diethylpyrocarbonate, kethoxal and carbodiimide. On the basis of these chemical data, a three-dimensional model was constructed by computer modeling and compared to that of canonical tRNA Ser, tRNA Sec resembles tRNA Ser at the level of its T-arm and anticodon-arm conformations, as well as at the joining of the D- and T-loops by a tertiary Watson-Crick G19-C56 interaction. Its extra-long variable arm is a double-stranded structure closed by a four nucleotide loop that is linked to the body of the tRNA in a way different from that found in tRNA Ser. As anticipated from the peculiar features of the sequence in the D-loop and at the junction of amino acid and D-arms, tRNA Sec possesses a novel but restricted set of tertiary interactions in the core of its three-dimensional structure: a G8-A21-U14 triple pair and a novel interaction between C16 of the D-loop and C59 of the T-loop. A third interaction involving C15-G20a-G48 is suggested but some experimental evidence for it is still lacking. It is furthermore concluded that the D-arm has six base-pairs instead of three, as in canonical II tRNA Ser, with the D-loop containing only four nucleotides. Finally, the amino acid accepting arm forms a stack of eight Watson-Crick base-pairs (instead of 7 in other tRNAs). The biological relevance of this model with regard to interaction with seryl-tRNA synthetase and enzymes from the selenocysteine metabolism is discussed.