The secondary structure of eukaryotic selenocysteine tRNA: 7/5 versus 9/4.

Insertion of selenocysteine into a growing peptide requires the unusual tRNASec (Zinoni et al+, 1987; Stadtman,1990;Böck et al+,1991)+This tRNAhas an extended D-stem containing six base pairs, which, in the case of eukaryotic tRNASec (euk-tRNASec), is the key identity element for selenylation and phosphorylation (Wu & Gross,1994;Amberg et al+,1996)+Two secondary structures have been proposed for the euk-tRNASec, which differ in the base pairing of the acceptor/T helical domain (Diamond et al+, 1981; Böck et al+, 1991; Sturchler et al+, 1993)+ One structure has the normal seven base pairs in the acceptor stem and five base pairs in the T-stem (7/5 structure, Fig+ 1, left), and is characterized by an unusually long four-nucleotide unpaired region between the acceptor and D-stems (Connector 1) and an unpaired nucleotide, C64a, in the T-stem+ The alternate structure features the normal two nucleotides in Connector 1 and a 13-base pair acceptor/T domain comprised of nine base pairs in the acceptor stem and four in the T-stem (9/4 structure, Fig+ 1, right)+ This 9/4 structure was initially proposed by analogy with the prokaryotic tRNASec (prok-tRNASec), which also contains 13 base pairs in the acceptor/T helical domain+However, in this case, there are eight and five base pairs in the acceptor and T-stems, respectively+ The acceptor/T helical domain having 13 base pairs is thought to be a key structural element determining the functionalities pattern of tRNASec in both prokaryotes and eukaryotes (Böck et al+, 1991)+ Using enzymatic and chemical probing, Sturchler et al+ (1993) favored the 9/4 structure, for which a threedimensional model was proposed+ Since then, new experimental data have been collected on serylation, selenylation, and phosphorylation of the euk-tRNASec and mutants thereof (Wu & Gross, 1993, 1994; Ohama et al+, 1994; Sturchler-Pierrat et al+, 1995; Amberg et al+, 1996)+ The point by point analysis presented here shows that the activities of the euk-tRNASec and its mutants in serylation, selenylation, and phosphorylation are better explained by the 7/5 structure+