Binding of misacylated tRNAs to the ribosomal A site

Abstract

To test whether the ribosome displays specificity for the esterified amino acid and the tRNA body of an aminoacyl-tRNA (aatRNA), the stabilities of 4 correctly acylated and 12 misacylated tRNAs in the ribosomal A site were determined. By introducing the GAC (valine) anticodon into each tRNA, a constant anticodonÁcodon interaction was maintained, thus removing concern that different anticodonÁcodon strengths might affect the binding of the different aa-tRNAs to the A site. Surprisingly, all 16 aatRNAs displayed similar dissociation rate constants from the A site. These results suggest that either the ribosome is not specific for different amino acids and tRNA bodies when intact aa-tRNAs are used or the specificity for the amino acid side chain and tRNA body is masked by a conformational change upon aa-tRNA release.Keywords: tRNA; ribosome; uniformity; misacylated tRNA; A site; ribosome binding Several studies indicate that the rate of incorporation of an amino acid into a growing polypeptide chain in Escherichia coli is roughly uniform when the substrate, aa-tRNAÁEF-TuÁGTP ternary complex, is saturating. For example, Curran and Yarus (1989) evaluated 18 elongator aminoacyl-tRNAs (aa-tRNAs) in their ability to compete with a frameshift and concluded that the rates of incorporation of aa-tRNAs belonging to codons used frequently in highly expressed genes were all near the average aa-tRNA selection rate, while aa-tRNAs that were not preferred in highly expressed genes were incorporated faster or slower than the mean. This observation supports the proposal made by Grosjean and Fiers (1982), who suggested that codons used in highly expressed genes are selected for maintenance of a uniform anticodonÁcodon interaction strength. Furthermore, Pedersen (1984) found that, in three different growth media, the translation rates of several different mRNAs varied by less than twofold, and that the small differences observed were due to the low concentration of aa-tRNAs belonging to rare codons. Thus, while the lower concentration of rare aatRNAs (and thus ternary complexes) can lead to slower translation rates, it appears that all aa-tRNAs are used in an equivalent manner during the translation of highly expressed genes.Not only does the overall rate of translation appear to be uniform, but there is accumulating biochemical evidence that several of the individual steps of translation elongation also display uniformity. It has been shown that the GTPbound form of elongation factor Tu (EF-TuÁGTP) binds all elongator aa-tRNAs with similar affinities (Louie et al. 1984;Ott et al. 1990). In addition, recent studies by have demonstrated that eight different purified aa-tRNAs bind the ribosomal A and P sites with a very small range of K D values. Finally, kinetic studies have shown that Phe-tRNA Phe and Trp-tRNA Trp have similar rates of GTP hydrolysis and peptide bond formation (Pape et al. 1998;Cochella and Green 2005).The manner in which uniformity is achieved is difficult to understand given the substantial diversity amon...

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

“…As shown in Figure 5, the ΔG° values of the tRNA Phe chimeras correlated closely to the ΔG° values obtained previously for the valylated E. coli tRNAs in nearly identical buffer conditions 6. The difference in the absolute value of ΔG° of the two data sets is primarily accounted for by the fact that tRNAs esterified with valine bind EF-Tu more weakly than the same tRNAs esterified with phenylalanine 19,20. However, the slope of the best fit line for the seven data points is 1.4, suggesting that some aspect of the bodies of several of the tRNAs outside of base pairs 49-65, 50-64, and 51-63 may also contribute to the binding affinity.…”

Phenylalanyl-tRNA synthetase editing defects result in efficient mistranslation of phenylalanine codons as tyrosine

“…3B), confirming that the ribosome lacks discrimination between Tyr and Phe side chains in the context of tRNA Phe . Previous studies showed that the ribosomal A site has different affinities for various aminoacyl-dinucleotide and puromycin derivatives (Bhuta et al 1981;Starck et al 2003), while nonenzymatic binding experiments suggested that the A site binds all tested misacylated tRNAs and cognate aa-tRNAs uniformly (Fahlman et al 2004;Dale and Uhlenbeck 2005b). Upon codon-anticodon recognition, the ribosome undergoes a series of conformational changes (Pape et al 1998(Pape et al , 1999(Pape et al , 2000Ogle et al 2002;Blanchard et al 2004;Gromadski and Rodnina 2004a,b;Cochella and Green 2005).…”

“…Translation inhibition experiments using various puromycin derivatives also indicated that the ribosomal A site specifically recognizes the amino acid side chains of aa-tRNAs (Starck et al 2003). Conversely, nonenzymatic binding studies using full-length aa-tRNAs showed that misacylated and correctly acylated tRNAs bind to the ribosomal A site with similar affinities (Fahlman et al 2004;Dale and Uhlenbeck 2005b). To provide some insights into the discrepancy, we investigated the effect on decoding kinetics of full-length misacylated tRNAs, as part of a broader study of the quality control mechanisms that prevent misincorporation of Tyr at Phe codons.…”

[3′-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation

“…tRNAs radiolabeled by this protocol have been used to examine many different aspects of protein synthesis. [3'-32 P] tRNAs have been used to examine the interactions between tRNA and modification enzymes [27], the kinetics of tRNA binding to the ribosome [28][29][30][31][32], and the "structure mapping" of tRNAs bound to the ribosome [33].…”

“…This assay is especially beneficial for the study of non-natural amino acids since radioactive derivatives are generally not available [10]. Our laboratory has used this method with 13 different aaRSs with either cognate or non-cognate tRNAs [8,30,32] (Ledoux and Uhlenbeck, submitted). Other groups have reported that 15 of the 20 canonical amino acids, as well as pyrolysine, have been esterified to tRNAs using this approach (Table 1) [8,30,[34][35][36][37][39][40][41][42][43][44].…”