Antideterminants present in minihelixSec hinder its recognition by prokaryotic elongation factor Tu

Abstract

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNASec form ternary complexes with activated elongation factor. tRNASec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNASec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNAAsp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNASec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNASec in which sequence elements of tRNAAsp were introduced showed that the sequence of the amino acid-acceptor branch of Escherichia coli tRNASec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNASec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7·G66/G49·U65/ C50·G64 box was tested by its transplantation into a minihelix derived from tRNAAsp, abolishing its recognition by EF-Tu·GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.