Mechanistics of the binding, function and\ud dissociation of ribosomal ligands during translation\ud initiation analysed by fast kinetics\ud

Initiation is the most regulated and rate limiting step of mRNA translation. In prokaryotes translation begins with an initial interaction between 30S ribosomal subunit and initiation factors (IF1, IF2, IF3), fMet-tRNAfMet and mRNA to eventually yield a 30S initiation complex (30S IC) which subsequently interacts with the 50S subunit to produce a 70S initiation complex (70S IC). Although biochemical and\ud structural properties of most of these interactions have been extensively characterized in the past, there are fundamental aspects of the kinetics of initiation\ud factors association to and dissociation from the ribosomal subunits which have not been extensively and/or directly studied and which therefore remain somewhat\ud controversial. In the present work, I present a direct study of the kinetics of binding and release of IF1 and IF3 using florescent reporter groups on each ribosomal ligand\ud involved in translation initiation (Chapter 1).\ud The functional implications and properties of the nucleotide binding domain of IF2 have been investigated. It is shown that translation initiation may be regulated by\ud switching the IF2 conformation from an active GTP-bound form to an inactive ppGppbound form. Thus, the guanine nucleotide binding domain confers upon IF2 the\ud properties of a metabolic sensor and might explain its evolutionary conservation (Chapter 2).\ud \ud \ud \u