Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu fromE.coli

Abstract

Selenocysteine-incorporating tRNA^Sec(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine. The equilibrium dissociation constant for the interaction of Ser- tRNA^Sec(UCA) with elongation factor Tu GTP was determined to be 5.0±2.5×10⁻⁸ M. Compared with the dissociation constants of the two elongator Ser-tRNA^Ser species (K_d = 7 × 10⁻¹⁰ M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu·GTP. This suggests a mechanism by which the Ser-tRNA^Sec is prevented in recognition of UGA codons. This tRNA is not bound to EF-Tu·GTP and is converted to selenocysteinyl-tRNA^Sec. We also demonstrate the lack of an efficient interaction of Sec with EF-Tu·GTP. The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu·GTP.