Escherichia colielongation‐factor‐Tu mutants with decreased affinity for aminoacyl‐tRNA

Abstract

The two evolutionary well-conserved histidine residues, His66 and His11 8, of Escherichia coli elongation factor Tu have been subjected to mutational analysis. The two histidines have each been replaced by alanines, denoted H66A and H11 8A, respectively. His118 has also been substituted by glutamate, H11 SE. The three mutants have been characterized with respect to thermostability, GTPase activity and affinity for aminoacylated tRNA. Most conspicuously, the tRNA affinity is reduced or almost abolished. k-, for dissociation of the ternary complex increases by factors of 14, 40 and 48 for H66A, H118A and H118E, respectively, when compared to the wild type. The halflives for the non-enzymic deacylation of aminoacylated tRNA in the ternary complex are 391, 107, 69, 54 and 61 min for wild type, H66A, H118A, H118E and free aminoacylated tRNA, respectively. The Kd is about 20-times higher for H66A compared to wild type.Our results strongly suggest that His66 and His118 play major roles in stabilization of the ternary complex.Elongation factor Tu (EF-Tu) from Escherichia coli is a monomeric protein consisting of 393 amino acid residues, and with a molecular mass of 43 kDa. During the elongation step in protein biosynthesis EF-Tu promotes the binding of aminoacyl-tRNA to the A-site of the programmed ribosome in the process of decoding the mRNA [l]. In its active conformation with GTP as a cofactor, EF-Tu has a high affinity for aminoacyl-tRNA with equilibrium dissociation constants in the range of 10-'M [2], the affinity for non-acylated tRNA being several orders of magnitudes lower [3].The low intrinsic GTPase activity of EF-Tu is stimulated on the ribosome when EF-Tu is present in the form of a ternary complex with GTP and aminoacyl-tRNA. Upon GTP hydrolysis EF-Tu undergoes a conformational change to a GDP form with negligible affinity for aminoacyl-tRNA. The exchange factor EF-Ts catalyses the replacement of GDP with GTP and thus completes the cycle.The crystal structure of E. coli EF-Tu in complex with GDP has been determined at high resolution [4] and so has the structure of EF-Tu :GTP from the thermophilic bacteria Thermus aquaticus and Thermus thermophilus [5, 61. Theoretical speculations about the biochemical nature of the important intermediate in the elongation process, the ternary complex, has brought about many interesting studies over the last two decades [7, 81. No unequivocal determination of macromolecular recognitory interaction sites has so far been established. To map the interaction points of EF-Tu and aminoacyltRNA different chemical modification studies have been applied in addition to cross-linking and site-directed mutagenesis studies.Modification studies of Cys81 indicated that this residue took part in tRNA binding [9, 101. This conclusion could not, however, be confirmed by mutagenesis studies [11].In three different studies [12-141 several lysine and arginine residues have been indicated to be involved in tRNA binding, in particular Lys2, Lys4, and Lys263. As EF-Tu is expected to recognize...

Histidine 66 in Escherichia coli Elongation Factor Tu Selectively Stabilizes Aminoacyl-tRNAs

“…ticipates in stabilizing many of these aa-tRNAs. The destabilization observed for Phe-tRNA Phe is similar to the value measured previously using a different assay (16).…”

“…This suggests that the loss of the stacking energy between the aminoacyl phenylalanine and His-66 does not dramatically affect the rates of accommodation or peptide bond formation on the ribosome. Interestingly, despite a reduced affinity of H66A EF-Tu for Phe-tRNA Phe off the ribosome, only a slight impact was observed in k pep similar to what had been previously observed in polyphenylalanine synthesis (16).…”

“…Although the position of His-66 does not change substantially during initial binding, substantial structural rearrangements occur in nearby regions of both the tRNA and EF-Tu upon GTP hydrolysis (13,14), suggesting that the H66A mutation may affect the kinetics of decoding. Although earlier experiments indicated that the H66A mutation had little effect on polyphenylalanine synthesis (16), this steady state assay could potentially obscure a kinetic effect in one of the sub-steps in decoding.…”

“…It is known that the H66A and H66L mutations of E. coli EF-Tu destabilize the binding to Phe-tRNA Phe (16,17), suggesting that the imidazole side chain can stabilize the esterified Phe. Here we test how the H66A mutation affects the binding of tRNAs esterified with other amino acids and how other mutations of His-66 can affect the specificity for aa-tRNAs.…”

Mutants of EF‐Tu defective in binding aminoacyl‐tRNA

“…It is known from previous work that substitutions of the conserved residue His 118 to Ala or to Glu [9] or to Gly [10] in E. coli EF-Tu also cause significant decreases in the affinity of EF-Tu for aa-tRNA. His us is in the immediate structural neighbourhood of Leu 12° and Gin 124 in the interface of domains I and III.…”