Mechanism of ribosomal recruitment of initiator tRNA in bacteria

Initiation of mRNA translation in prokaryotes requires the small ribosomal subunit (30S), three initiation factors, IF1, IF2 and IF3, initiator tRNA (fMet-tRNAfMet) and the large ribosomal subunit (50S). During initiation, the 30S ribosomal subunit interacts with the three factors and binds in a presumably random manner fMet-tRNAfMet and mRNA. This initially formed 30S pre-initiation complex is a kinetic intermediate of the stable 30S initiation complex which is formed upon base pairing of the anticodon of fMet-tRNAfMet and the intiation codon of the mRNA in the P-site. In a subsequent step the 50S subunit binds to the 30S initiation complex giving rise to a 70S initiation complex. During this step, a molecule of GTP bound to IF2 is hydrolyzed, the initiation factors are released from the ribosome and fMet-tRNAfMet is stabilized in the P site ready to enter in the elongation phase of translation. In turn, each of these major steps involves numerous elemental steps, whose sequence and timing are largely unknown. How the fMettRNAfMet is bound to the ribosome during the assembly of the initiation complex is one of the many questions still remaining open. According to the classical model, IF2 acts as a carrier of fMet-tRNAfMet to the ribosome. An alternative model predicts that the IF2, prebound to the 30S subunit awaits the arrival of fMet-tRNAfMet with which it interacts only at the ribosomal surface. This work presents results of a kinetic analysis of binding to the 30S subunit of various ribosomal ligands (Initiation Factors, fMet-tRNAfMet, mRNA), performed with fast kinetic techniques (rapid filter binding, fluorescence stopped flow). The data obtained strongly support the model in which IF2 is not a fMettRNAfMet carrier. The elemental rate constants determined here give us a more detailed description of the process of 30S initiation complex assembly, of the interplay of the various ribosomal ligands and of the functional significance of different structural modules of IF2