The Mechanism of translation termination in Escherichia coli

Abstract: Protein synthesis in E. coli is terminated by the release factors (RFs) and the ribosome is prepared for another round of protein synthesis by ribosome recycling factor (RRF), elongation factor G (EF-G) and initiation factor (IF3). In this thesis, the mechanism and specificity of RF1 and RF2 and the role of RRF and RF3 in termination were studied using biochemical assays.The release factors RF1 and RF2 recognize the stop codons (RF1 acts at UAG and UAA codons and RF2 at UAA and UGA) and trigger the hydrolysis of peptidyl-tRNA, which results in the release of the protein from the ribosome. Then, RF3 dissociates RFI/RF2 from the ribosome in a reaction that involves the hydrolysis of GTP. RF3 does not affect the effective association rate (kcat/KM) or the catalytic rate (kcat) of peptidyl-tRNA hydrolysis by RF1 or RF2. The sole effect of RF3 is, therefore, to remove RF1 and RF2 after release of the nascent peptide.RF3 and RRF are required for the fast recycling of ribosomes. In the absence of RF3, increasing the concentrations of RF1 or RF2 caused inhibition of ribosome recycling that could be reversed by corresponding increases in RRF concentration indicating that RF1/RF2 and RRF have mutually exclusive binding sites on the ribosome. The inhibition of ribosome recycling by RF1/RF2 was dependent on the nucleotide base immediately following the stop codon, which indicates that the affinity of the release factors to the ribosome is sensitive to the context of the stop codon. This was confirmed in direct measurements, where the relative kcat/KM values for RF1 and RF2 were measured for different stop codon contexts.The recognition of the stop codon by RF1 and RF2 is highly accurate. The normalized accuracy (fold change in the (kcat/KM) was measured for all codons that are different from a stop codon by one nucleotide. Accuracy values ranged from 103 to more than 106. This means that the reading of the codon by the release factors is the most accurate protein-RNA interaction known. The accuracy of stop codon recognition was not affected by aminoglycoside antibiotics, while the accuracy of codon recognition by aminoacyl-tRNA was reduced by several orders of magnitude in the presence of these antibiotics.