Quantitative Assessment of EF-1α·GTP Binding to Aminoacyl-tRNAs, Aminoacyl-viral RNA, and tRNA Shows Close Correspondence to the RNA Binding Properties of EF-Tu

Theo W Dreher,Olke C Uhlenbeck,Karen S Browning

doi:10.1074/jbc.274.2.666

Abstract

A ribonuclease protection assay was used to determine the equilibrium dissociation constants (Kd) for the binding of various RNAs by wheat germ EF-1alpha.GTP. Aminoacylated fully modified tRNAs and unmodified tRNA transcripts of four specificities (valyl, methionyl, alanyl, and phenylalanyl) from higher plants or Escherichia coli were bound with Kd values between 0.8 and 10 nM. A valylated 3'-fragment of turnip yellow mosaic virus RNA, which has a pseudoknotted amino acid acceptor stem, was bound with affinity similar to that of Val-tRNAVal. Uncharged tRNA and initiator Met-tRNAMet from wheat germ, RNAs that are normally excluded from the ribosomal A site in vivo, bound weakly. The discrimination against wheat germ initiator Met-tRNAMet was almost entirely due to the 2'-phosphoribosyl modification at nucleotide G64, since removal resulted in tight binding by EF-1alpha.GTP. A 44-nucleotide RNA representing a kinked acceptor/T arm obtained by in vitro selection to bacterial EF-Tu formed an Ala-RNA.EF-1alpha.GTP complex with a Kd 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-1alpha (eEF1A) therefore closely resembles that of bacterial EF-Tu (EF1A).

Highlights

A ribonuclease protection assay was used to determine the equilibrium dissociation constants (Kd) for the binding of various RNAs by wheat germ EF-1␣1⁄7GTP
High Affinity Binding of EF-1␣1⁄7GTP to Aminoacylated tRNAs—The equilibrium dissociation constants of ternary complexes formed by wheat germ EF-1␣1⁄7GTP with tRNAs of four specificities were between 0.8 and 10 nM (Table I), indicating a high affinity interaction similar to that between E. coli EF-Tu1⁄7GTP and aminoacyl-tRNAs (Kd values of 0.2–7 nM; Refs. 1–3)
Phenylalanyl-tRNAPhe from E. coli was bound by EF-1␣1⁄7GTP with the weakest affinity of the aminoacyl-tRNAs tested

Summary

EXPERIMENTAL PROCEDURES

Preparation of RNAs—Mature tRNAPhe from E. coli strain MRE600 was purchased from Boehringer Mannheim, total wheat germ tRNA was purchased from Sigma, and purified yeast initiator tRNAMet was a gift from Drs C. Counts (cpm) from direct counting in scintillation fluid at known efficiency were compared with counts obtained after taking an aliquot of the labeled tRNA in 20 ␮l of binding buffer through a mock assay termination procedure (including addition of RNA and spotting onto filter paper). This specific activity measurement included adjustment for losses during transfer to the filter paper and from incomplete precipitation onto the paper. The treated tRNAs were methionylated by a wheat germ methionyl-tRNA synthetase activity in the presence of (CTP,ATP):tRNA nucleotidyltransferase, which is able to regenerate the eliminated 3Ј-A residue

RESULTS

Ala minihelix

DISCUSSION