Role of the Initiation Factors in mRNA Start Site Selection and fMet-tRNA Recruitment by Bacterial Ribosomes

Translation initiation is a complex, multi-step process of fundamental importance in all kingdoms of life, during which the start site of the genetic message transmitted in the form of an RNA molecule (mRNA) is selected, and the level of translation determined. Being the slowest step of protein synthesis, initiation is the phase most often subject to regulation. Here we review, in a historical perspective and focusing mainly on results from our laboratory, the development of our perception of the mechanisms by which the most relevant steps of this pathway occur in bacteria. In particular, we describe: (a) the mechanistics and kinetics of translation initiation; (b) properties of mRNAs with and without Shine–Dalgarno sequence relevant for initiation site selection and translational efficiency; c) ribosomal binding and dissociation of the initiation factors, formation and properties of translation initiation intermediates; (d) the mechanisms by which translation initiation fidelity is ensured. Finally, we provide a short survey of the known translation initiation inhibitors.     

Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acids involved in its ribosomal binding and recycling

Starting from a synthetic modular gene (infA^*) encoding Escherichiacoli translation initiation factor IF1, we have constructedmutants in which amino acids are deleted from the carboxyl terminusor in which His29 or His34 are replaced by Tyr or Asp residues.The mutant proteins were overproduced, purified and tested invitro for their properties in several partial reactions of thetranslation initiation pathway and for their capacity to stimulateMS2 RNA-dependent protein synthesis. The results allow for theconclusion that: (i) Arg69 is part of the 30S ribosomal subunitbinding site of IF1 and its deletion results in the substantialloss of all IF1 functions; (ii) neither one of its two histidinesis essential for the binding of IF1 to the 30S ribosomal subunit,for the stimulation of fMet-tRNA binding to 30S or 70S ribosomalparticles or for MS2 RNA-dependent protein synthesis; but (iii)His29 is involved in the 50S subunit-induced ejection of IF1from the 30S ribosomal subunit.