Initiation of protein synthesis in Escherichia coli is a catalytic process, provided that conditions for hydrolysis of GTP are present. This means that the initiation factors IF-1, IF-2 and IF-3 are released at a certain stage of initiation complex formation, whereafter they recycle and are reutilized in another round of initiation.Experiments presented in this paper provide evidence for the mechanism of recycling, in which the hydrolysis of GTP and IF-1 both play a predominant role. We demonstrate that binding of met-tRNA, which is dependent on IF-2, is a stoichiometric process in a system devoid of IF-1. Addition of IF-1 converts it into a catalytic process, however, only if GTP can be hydrolysed. No such conversion takes place with 5'-guanylylmethylenediphosphonate. The same observation is valid for the uncoupled IF-2 dependent GTPase activity.I n the process of initiation IF-1 acts as a recycling promoting factor, which displays analogous properties to elongation factor EF-Ts. We postulate that a ribosomal-bound IF-2 * GDP complex is converted into an IF-2 -GTP complex, which is released and is capable of starting a new cycle of initiation.Furthermore, kinetic evidence is presented that IF-3 recycles even a t the level of 30-5 initiation complex formation, irrespective of whether conditions for hydrolysis of CTP are provided.The formation of an initiation complex, containing an Escherichia coli 70-5 ribosome, bacteriophage MS2-RNA and formylmethionyl-tRNA, is mediated by the initiation factors IF-1, IF-2 and IF-3 [l-31. The junction of the 50-5 subunit to the 30-5 initiation complex triggers a number of successive reactions which finally lead to release of the factors involved in the formation of the complex and to hydrolysis of GTP into GDP and inorganic phosphate. We have focussed our attention on the successive events in initiation in order to establish the separate functions of the initiation factors. The release of these factors, coupled with hydrolysis of GTP and their subsequent reutilization in another round of initiation, designated as recycling, covers the main part of this investigation. Enzynwr. Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); phosphoglycerate kinase (EC 2.7.2.3).Definition. Aezo unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 620 (260)nm, when measured in 8 1-cm-pathlength cell.It is known that IF-2 resembles very closely the elongation fa,ctor EF-Tu [4,5]. A ternary complex of IF-2 GTP * met-tRNA mediates the binding of met-tRNA to a 3043 MS2-RNA complex. IF-3 promotes the binding of natural messenger to 30-5 ribosomal subunits. However, the mechanism through which IF-3 is capable of promoting this binding has remained unclear [6-91. The function of IF-1 has been assumed to be a protein factor for the stabilization of the initiation complex [lo]. We have extended, however, the analogy with elongation factors and proposed that IF-1 is a recycling promoting factor, which displays analogous properties to EF-Ts Ell].The use of 8...