Kinetics of initiation of bacterial protein synthesis

Abstract

The 30S initiation complex, formed with the 30S ribosomal subunit, mRNA, and fMet-tRNA, has been shown by kinetic analysis with limiting concentrations of Escherichia coli ribosomes to be an obligatory intermediate in the formation of the 70S initiation complex. The formation of the 70S initiation complex began with an induction period and was proportional to the concentration of the 30S complex, which rapidly rose to a peak. The entire time course of the sequential pseudo-first-order, second-order reaction was reproduced accurately by the overall rate expression, in which we used rate constants that were determined by carrying out 30S and 70S complex formation separately. By using limiting concentrations of mRNA, we showed that phage MS2 RNA contained no specific signal that enhanced its rate of 30S complex formation with E. coli ribosomes and initiation factors; the pseudo-first-order rate constants obtained with poly(A3C9G1U1), poly(C15G1U4), and poly(G1U3) were 12-45 times higher than that with MS2 RNA. The observation that the rate constants for binding of fMet-tRNA and AcPhe-tRNA with a given synthetic RNA were comparable indicated that the initiator codon is recognized only indirectly through the initiator tRNA.