Choice of tRNA on Translating Ribosomes

Sammanfattning: This thesis addresses different aspects of the question about accuracy of protein synthesis: i) the mechanism of tRNA selection during translation ii) study of ribosomal mutations that affect accuracy and iii) the choice of aminoacyl-tRNA isoacceptors on synonymous codons.By measuring the codon reading efficiencies of cognate and near-cognate ternary complexes we demonstrate that in optimal physiological conditions accuracy of substrate selection is much higher than previously reported; that during translation the ribosomal A site is not blocked by unspecific binding of the non-cognate tRNAs which would inhibit the speed of protein synthesis. Our results suggest that there is an asymmetry between initial selection and proofreading step concerning the wobble position, and that binding of non-cognate substrate does not induce GTP hydrolysis on the ribosome.The knowledge obtained from the ribosomal mutant strains can be used to explain the general relation between the structure of the ribosome and the mechanism of codon recognition, as well as the streptomycin resistance or dependence phenomenon.Our work showed experimentally that the probability for binding certain tRNA to the A site of the ribosome is not based on the simple codon-anticodon base pair matching. In the living cell the availability of cognate tRNAs versus the demand for them (the frequency of codon usage) is finely balanced to ensure critical protein synthesis in stress conditions. We have also discovered a new codon assignment for a specific tRNALeu isoacceptor and detected a base modification in its anticodon, which has not been previously observed. The motivation for the later findings comes from a system biology modeling and the results are an example of an interdisciplinary collaboration.