PHYSIOLOGICAL-EFFECTS OF TRANSLATION INITIATION-FACTOR IF3 AND RIBOSOMAL-PROTEIN L20 LIMITATION IN ESCHERICHIA-COLI

To investigate the physiological roles of translation initiation facto r IF3 and ribosomal protein L20 in Escherichia coli, the infC, rpmI an d rpIT genes encoding IF3, L35 and L20, respectively, were placed unde r the control of Inc promoter-/operator sequences. Thus, their express ion is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombin ant lambda phages that express either rpmI and rplT or infC and rpmI i n trans, thereby allowing depletion of only IF3 or L20 at low IPTG con centrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo . During slow growth, the ratio of RNA to protein increases rather tha n decreases as occurs with control strains, indicating that IF3 limita tion disrupts feedback inhibition of rRNA synthesis. As IF3 levels dro p: expression from an AUU-infC-lacZ fusion increases, whereas expressi on decreases from an AUG-infC-lacZ fusion, thereby confirming the mode l of autogenous regulation of infC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decreas e in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41-43S is seen. Previou s studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepr esses the expression of an rplT-lacZ gene fusion, thus confirming auto genous regulation by L20.