Quantitative assessment of EF-1 alpha center dot GTP binding to aminoacyl-tRNAs, aminoacyl-viral RNA, and tRNA shows close correspondence to the RNA binding properties of EF-Tu

A ribonuclease protection assay was used to determine the equilibrium disso ciation constants (K-d) for the binding of various RNAs by wheat germ EF-1 alpha.GTP. Aminoacylated fully modified tRNAs and unmodified tRNA transcrip ts of four specificities (valyl, methionyl, alanyl, and phenylalanyl) hom h igher plants or Escherichia coil were bound with K-d values between 0.8 and 10 nM. A valylated 3'-fragment of turnip yellow mosaic virus RNA, which ha s a pseudoknotted amino acid acceptor stem, was bound with affinity similar to that of Val-tRNA(Val). Uncharged tRNA and initiator Met-tRNA(Met) from wheat germ, RNAs that are normally excluded from the ribosomal A site in vi vo, bound weakly. The discrimination against wheat germ initiator Met-tRNA( Met) was almost entirely due to the 2'-phosphoribosyl modification at nucle otide G64, since removal resulted in tight binding by EF-1 alpha.GTP. A 44- nucleotide RNA representing a kinked acceptor/T arm obtained by in vitro se lection to bacterial EF-Tu formed an Ala-RNA EF1 alpha.GTP complex with a K -d of 29 nM, indicating that much of the binding affinity for aminoacylated tRNA is derived from interaction with the acceptor/T half of the molecule. The pattern of tRNA interaction observed for EF-1 alpha (eEF1A) therefore closely resembles that of bacterial EF-Tu (EF1A).