DEFINING A SMALLER RNA SUBSTRATE FOR ELONGATION-FACTOR TU

A nuclease protection assay was used to obtain equilibrium dissociatio n constants of Thermus thermophilus EF-Tu with two well-characterized internal deletions of Escherichia coli Ala-tRNA(Ala) and yeast Phe-tRN A(Phe). Aminoacylated tRNAs with the anticodon hairpin substituted by a tetranucleotide bind to EF-Tu as well as the corresponding full-size d tRNAs. However, the Ala minihelix, where residue A7 is joined direct ly to A49, binds to EF-Tu less well than the full-sized Ala-tRNA(Ala). Similar data were obtained for Escherichia coli EF-Tu. An in vitro se lection strategy was used to isolate a substrate for EF-Tu from an RNA library where nine random nucleotides inserted between A7 and A49 in the Ala minihelix. After six rounds of enrichment, two groups of RNA w ere obtained that bound T, thermophilus EF-Tu as well as Ala-tRNA(Ala) . Group I molecules have the consensus sequence UNDUGACUY (N = U, C, A , G; D = U, G; Y = U, C) in the randomized region, and Group II molecu les generally have 5'-terminal GUG, but are more variable in the remai ning six nucleotides. The selected RNAs bind EF-Tu better than the min ihelix either because they provide additional function groups for prot ein binding or because they have a structure more similar to the amino acyl acceptor branch of tRNA.