Evidence for a guanine nucleotide-aminoacyl-RNA complex as an intermediate in the enzymatic transfer of aminoacyl-RNA to ribosomes

Recognition of the universally conserved 3′-CCA end of tRNA by elongation factor EF-Tu

“…The ternary complex of bacterial elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA plays a crucial role in translating the genetic code during protein synthesis by carrying activated amino acids to the A-site of messenger RNA-programmed ribosomes+ EF-Tu:GTP does not differentiate among the various aminoacylated elongator tRNAs, but recognizes nucleotide sequences or structural features common to all+ In formation of the ternary complex, EF-Tu:GTP discriminates against peptidyl-tRNAs and uncharged tRNAs (Gordon, 1967;Ravel et al+, 1967), which implicates the aminoacylated 39 end of tRNA as an important recognition site+ Biochemical and biophysical evidence suggests that other regions of tRNA are also involved in recognition by EF-Tu:GTP, including portions of the continuous acceptor stem/ T-stem helix and the variable loop of the tRNA (reviewed in Faulhammer & Joshi, 1987;Reinbolt et al+, 1993;Nissen et al+, 1996)+ The recently solved crystal structure of the ternary complex (Nissen et al+, 1995) provides high-resolution details of the protein-tRNA interactions+ Modifications at the universally conserved 39-CCA sequence are known to inhibit assembly of the ternary complex (Faulhammer & Joshi, 1987)+ It is, however, difficult to analyze the role of the three terminal nucleotides in formation of the ternary complex because alteration of this sequence usually results in loss of aminoacylation activity (Sprinzl & Cramer, 1979)+ We have recently shown that systematic mutation of all three bases of the 39-CCA terminus of Escherichia coli tRNA Val yields tRNAs with altered 39 ends that are readily aminoacylated (Horowitz et al+, 1993;Liu & Horowitz, 1994; also see Tamura et al+, 1994)+ Valine tRNAs terminating in either 39 cytosine or 39 uracil retain almost full aminoacylation activity; the tRNA Val variant with a 39-terminal guanine is also fully chargeable, but is a poorer substrate for valyl-tRNA synthetase+ Cytosines at positions 74 and 75 are also not essential for aminoacylation; only the G75 mutant is a poor valine acceptor (Liu & Horowitz, 1994)+ Experiments testing the activity of 39-terminal mutants of tRNA Val in in vitro polypeptide synthesis showed that the variant with a 39 G76 substitution is active+ However, mutant tRNAs terminating in 39 pyrimidines, which are readily aminoacylated, are unable to transfer valine into polypeptides (Liu & Horowitz, 1994) (Liu & Horowitz, 1994)+ The conserved C74 and C75 are also not essential for aminoacylation of tRNA Val (Liu & Horowitz, 1994)+ Replacing either of the terminal C residues yields tRNAs that remain good substrates for ValRS+ Exceptions are the G75 mutant and the U74U75 double mutant, which accept valine poorly; both tRNAs can, however, be aminoacylated at high ValRS concentrations (Liu & Horowitz, 1994 (Ofen...…”

Properties and regulation of the GTPase activities of elongation factors Tu and G, and of initiation factor 2

“…(2) first associated a GTPase activity with a nonribosomal fraction involved in protein synthesis, corresponding to elongation factor G (EF-G). Later on, it was shown that also the interaction with ribosomes of elongation factor Tu (EF-Tu) (3,4) and initiation factor 2 (IF-2) (5) is accompanied by the hydrolysis of GTP. The binding of this nucleotide and its consequent ",/-phosphate hydrolysis induce a specific sequence of conformational changes of the factors, directly related to their functions in protein synthesis.…”

Interaction of elongation factor Tu with 2'(3')-O-aminoacyloligonucleotides derived from the 3' terminus of aminoacyl-tRNA.

“…Purified EF-Tu-GDP was kindly provided by Dr. David L. Miller (Roche Institute of Molecular Biology). EF-T (a mixture of EF-Tu and EF-Tj) was prepared from the 150,000 X g supernatant of Escherichia coil MRE 600 (RNase 1-) cells (General Biochemicals) by ammonium sulfate fractionation and DEAE-Sephadex chromatography as described by Leder (9 (2,3,12). The actual EF-Tu is known to be easily deactivated in the absence of sulfhydryl reagents (14).…”