Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli.

Abstract

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 in Escherichia coli, the infC, rpmI and rpIT genes encoding IF3, L35 and L20, respectively, were placed under the control of lac promotor/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express either rpmI and rplT or infC and prmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low 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 than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation of infC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease 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. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of an rplT-lacZ gene fusion, thus confirming autogenous regulation by L20.