Development and validation of reagents for understanding tRNA:ribosome interactions one molecule at a time

Translation is responsible for the production of all proteins in a cell making it crucial to the survival of all organisms. Translation involves the decoding of an mRNA by ribosomes and tRNAs. Studying tRNA-ribosome interactions in detail is important for modelling protein synthesis, and this has applications in bioprocessing and in understanding gene regulation in diseases. This project has set out to develop a single molecule technique to image each step of the ribosome:tRNA interaction process. This will enable studying the rate at which translation occurs as well as further define the steps that characterise this process. I have designed and cloned a synthetic DNA sequence which can be used to initiate translation in in vitro reactions. By omitting leucine from the translation reaction, ribosomes can be arrested at a specific leucine codon in a state where a six histidine tag protrudes from the ribosomal exit channel. This should enable immobilisation of translating ribosomes on metal-affinity surfaces. The functionality of the synthetic sequence was demonstrated in a reticulocyte lysate system. A complementary detection system that allows localising individual ribosome:mRNA complexes is currently being developed.