Mutational analysis of translational activities in bacteria

Protein biosynthesis occurs on large macromolecular ribo-nucleoprotein complexes, the ribosomes, in a process termed translation. In prokaryotes transcription and translation are tightly coupled cellular processes; ribosomes initiate translation on mRNAs while they are still being transcribed. Translation initiation is the rate-limiting and most highly regulated step of the three phases of protein biosynthesis (initiation, elongation and termination). Protein synthesis begins with an initial binding to the 30S ribosomal subunit of the initiation factors: IF3, IF1 and IF2 (according to the presumed order in which they bind). The initiator fMet-tRNAfMet and mRNA bind stochastically to the ribosomal subunit without initially interacting with each other, yielding a ''30S pre-initiation complex'', which can either dissociate into its individual components or undergo a conformational change consisting of at least two first order rearrangements that promotes codon-anticodon interaction and formation of the more stable ''30S initiation complex'' [...]. The transition from pre-initiation to the bona fide 30S IC is kinetically controlled by the initiation factors [...] and entails a conformational transition of the ribosomal subunit and a shift of the mRNA from the ''standy-by'' to the P-decoding site position. The 30S initiation complex can then be fixed by its association with the 50S ribosomal subunit to form a ''70S initiation complex''. Joining of the 50S subunit with the 30S initiation complex causes a number of events: the activation of GTP hydrolysis by IF2 and a conformational change of this initiation factor; the dissociation of the initiation factors IF1 and IF3 and the stabilization of fMet-tRNAfMet in the P site [...]. The newly formed 70S initiation complex with fMettRNAfMet in the peptidyltransferase center of the 50S ribosomal subunit is now ready to enter the elongation phase of translation