A biomolecular purification system for biophysical studies of protein synthesis

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Abstract Our research group seeks to understand the assembly and mechanism of eukaryotic protein synthesis machinery in order to identify critical steps that contribute to the molecular mechanisms that drive and regulate the translation of mRNA into proteins. To achieve this we have developed experimental methods that combine (i) reconstitution of in vitro translation systems from highly purified components; (ii) fluorescent site-specific labelling of these components and validation that the labeled components are active; (iii) biophysical assays to determine the binding affinity and kinetics of assembly of these complexes and (iv) expression of reporter mRNA to determine the functional effects of these protein-nucleic acid interactions. Specifically we are examining the roles of eIF4G1 and DAP5 in eIF4E-independent translation of mRNAs expressed under stress conditions. We have developed a unique set of assays and combined our expertise with single molecule fluorescent measurements done in Prof. Ruben Gonzalez' lab to provide new insights into the mechanism and regulation of mRNA expressed under stress conditions. We are requesting an AKTA purification system to obtain high quality samples necessary for our experiments.