The Development of Novel In Vitro Binding Assays to Further Elucidate the Role of tRNAs in Protein Synthesis

Abstract

The accurate translation of the genetic information encoded by mRNA into proteins is critical for proper cellular function. Transfer ribonucleic acids (tRNAs) are essential biomolecular linkers that help maintain translational fidelity by accurately connecting genotype (mRNA) to phenotype (protein) during protein synthesis. The initial stages of ribosomal protein synthesis are controlled by aminoacyl-tRNA synthetases (AARSs). AARSs initiate protein synthesis by catalyzing the acylation of tRNAs to their cognate amino acids. The resulting aminoacyltRNAs (aa-tRNAs) are transported to the ribosome by the translation factor Elongation Factor (EF-Tu) to undergo peptide bond formation. The synergistic interplay among the protein translational machinery involved in this process is highly regulated and extremely accurate. In E. coli, the error rate for protein synthesis correlates to roughly 1 misincorporation per 104 codons translated (Husaain et al., 2006; Loftield & Vanderjagt, 1972; Ogle & Ramakrishnan, 2005; Rosenberger & Foskett, 1981; Roy & Ibba, 2006). The high level of stringency is the result of substrate discrimination that can occur at four different translation checkpoints: 1) amino acid activation, 2) aminoacylation, 3) aa-tRNA transport to the ribosome (via EF-Tu) and 4) initial binding of aa-tRNAs within the ribosome. The molecular recognition events mediated by AARSs in checkpoints 1 and 2 provide the highest level of stringency. In vitro studies have shown that AARSs possess a discrimination factor of 200 – 10,000 during amino acid activation; the stringency factor increases to > 10,000 during aminoacylation (Giege et al., 1998; Ling et al., 2009; Reynolds et al., 2010). EF-Tu binding interactions provide an additional, albeit lower, degree of stringency in step 3. Reports show that EF-Tu binds cognate aa-tRNAs with nearly identical affinities, while noncognate or misaclyated aa-tRNAs possess extremely broad EF-Tu binding affinity profiles (~ 700-fold difference between noncognate aa-tRNAs) (Dale & Uhlenbeck; 2005). It is presumed that these species are removed from the translation pathway by EF-Tu to further enhance fidelity. Despite the large amount of studies focused on defining the intricacies of translation, there are still a number of unanswered questions regarding the basic tenants of translation. Two intriguing questions are: i) do tRNAs play an active or passive role in the regulation of translation and ii) why is ribosomal protein synthesis limited to 20 amino acids? An interrelated question that we would like to address is: can in vitro binding assays be used to accurately define the mechanisms of cellular protein synthesis? This chapter focuses on current and the development of two in vitro binding assays that are designed to investigate the role of tRNA in the initial and intermediate stages of translation. The new assays offer rapid, sensitive and straightforward techniques to monitor the role of tRNA in: activation,