Defining a smaller RNA substrate for elongation factor Tu

A nuclease protection assay was used to obtain equilibrium dissociation constants of Thermus thermophilus EF-Tu with two well-characterized internal deletions of Escherichia coli Ala-tRNA(Ala) and yeast Phe-tRNA(Phe). Aminoacylated tRNAs with the anticodon hairpin substituted by a tetranucleotide bind to EF-Tu as well as the corresponding full-sized tRNAs. However, the Ala minihelix, where residue A7 is joined directly 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 selection 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 were 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 molecules generally have 5'-terminal GUG, but are more variable in the remaining six nucleotides. The selected RNAs bind EF-Tu better than the minihelix either because they provide additional function groups for protein binding or because they have a structure more similar to the aminoacyl acceptor branch of tRNA.