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

Abstract

The translational elongation factors EF-1␣ (eEF1A) 1 and EF-Tu (EF1A) are GTP-binding proteins that serve similar roles in protein synthesis in prokaryotes and eukaryotes, respectively. Their normal role is to deliver aminoacylated tRNAs into the A site of the ribosome. While dispensing this function for tRNAs carrying all the standard 20 amino acids (including methionine) that are elongationally inserted into proteins, elongation factors must discriminate against non-aminoacylated tRNAs and against the methionylated initiator tRNA Met that is loaded into the ribosomal P site by initiation factors.The basis for these functions is well understood in bacterial systems, for which extensive ligand binding and structural studies have been performed. The reported equilibrium dissociation constants for the interactions of Escherichia coli EFTu⅐GTP with aminoacylated elongator tRNAs fall between 0.2 and 7 nM (1-3), varying over a 13.8-fold (4) or a 34-fold (2) range when determined in a single set of experiments. These tight associations forming aminoacyl-tRNA⅐EF-Tu⅐GTP ternary complexes contrast strongly with the weak affinity of E. coli EF-Tu⅐GTP for uncharged tRNA (K d ϭ 2.6 -2. Met from the A site thus relies only partially on EFTu⅐GTP discrimination, but also on A site competition by elongator methionyl-tRNA Met ⅐EF-Tu⅐GTP ternary complex, complex formation with IF2⅐GTP, and selective interaction of initiator tRNA with the ribosomal P site (5).The recently solved crystal structure of the phenylalanyltRNA Phe ⅐EF-Tu⅐GTP complex from Thermus aquaticus (6) has shown that the protein contacts the aminoacyl-tRNA only in the acceptor/T half of the tRNA molecule. Accordingly, aminoacylated tRNA half-molecules and tRNAs lacking the anticodon domain have been reported to bind Thermus thermophilus EF-Tu⅐GTP with affinities similar to those of the parental tRNAs (7,8).The overall similarity of EF-1␣ function to that of EF-Tu (see, e.g., Refs. 9 -11) has been convincingly established by the interchangeability of EF-Tu and EF-1␣ in binding to, although not in supporting protein synthesis by, bacterial and mammalian ribosomes (12). EF-1␣, like EF-Tu, has been shown to form ternary complexes with GTP and aminoacyl-tRNA (9, 10, 13). However, in contrast to the rich information available for EFTu, quantitative data on the interaction of EF-1␣ with tRNAs are almost non-existent. The only estimate for the stability of aminoacyl-tRNA⅐EF-1␣⅐GTP ternary complex we have found in the literature is one of "about 10 nM" for Phe-tRNA (11), and the extent of binding discrimination against uncharged tRNA is not known. With regard to the exclusion of initiator methionyltRNA Met from the A site of eukaryotic ribosomes, it has been shown that a 2Ј-phosphoribosyl modification of the purine at position 64 of the yeast and wheat germ initiator tRNAs is an important antideterminant of interaction with 15). After removal of this modification, both tRNAs could serve as elongators in in vitro protein synthesis (15); in the yeast case, the demodified met...

Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition

“…Starck and R.W. Roberts nanomolar affinity (K d ϭ 29 and 84 nM, respectively; Dreher et al+, 1999)+ The three differences between these compounds and our analogs are (1) the amide linkage attaching the amino acid to the ribose, (2) the o-methyl tyrosine side chain, and (3) the N6-dimethyl moiety+ The side chain likely has little effect on elongation factor binding, as many unnatural hydrophobic amino acids may be inserted into proteins in both bacteria (Ellman et al+, 1991) and in eukaryotes (Dougherty, 2000)+ The amide linkage and N6-dimethyl adenosine in our tRNA mimics may result in somewhat reduced affinity for the elongation factor (Baksht et al+, 1976)+ We presently favor a model where RNA 12-P and Ala-minihelix-P act by sequestering eEF1A for two reasons: (1) inhibition occurs as the concentration of RNA-12-P or Ala-minihelix-P becomes comparable to the endogenous eEF1A concentration (;20 mM) and (2) this concentration is approximately 1,000-fold higher than the reported K d s for alanyl-RNA 12 and -Alaminihelix with eEF1A (Nazarenko & Uhlenbeck, 1995;Dreher et al+, 1999)+ In this scenario, addition of minihelix titrates away the available pool of elongation factor until translation is entirely shut down+…”

Moonlighting Functions of Polypeptide Elongation Factor 1: From Actin Bundling to Zinc Finger Protein R1-Associated Nuclear Localization

“…Furthermore, the valyltRNA-like structure associates with EF-1a with an a‹nity similar to that of val-tRNA val . 115) Recently, a 5? cloverleaf structure in poliovirus RNA (positivestrand) has been shown to be a cis-acting replication element required for negative-strand synthesis.…”

Cap- and initiator tRNA-dependent initiation of TYMV polyprotein synthesis by ribosomes: Evaluation of the Trojan horse model for TYMV RNA translation

“…To study valine trans-incorporation into protein, TYMV virion RNA (1 mg; z0.5 pmol) or Brome mosaic virus (BMV) virion RNA (Promega, 0.25 mg) was incubated with [ (Dreher et al 1999). …”