Mechanism of the Inhibition of Protein Synthesis by Kirromycin. Role of Elongation Factor Tu and Ribosomes

Abstract

We have investigated the mechanism which allows the antibiotic kirromycin to inhibit peptide bond formation by acting on elongation factor Tu (EF-Tu). Gel filtration experiments show that with kirromycin after enzymic binding of aminoacyl-tRNA to ribosomes and subsequent GTP hydrolysis EF-Tu is not released from the ribosome. However, ability of the ribosomal peptidyl transferase center to support puromycin reactivity of peptidyl-tRNA bound to the ribosomal donor site is still preserved. This shows that inhibition of peptide bond formation by kirromycin is the consequence of the failure of EF-Tu to leave the ribosome, which blocks interaction of the C-C-A end of aminoacyl-tRNA bound to the acceptor site with the peptityl-transferase center. In line with these observations, kirromycin inhibits ribosome · EF-Tu GTPase activity down to one round of GTP hydrolysis when poly(U) and Phe-tRNAPhe are simultaneously present. The intrinsic inability of EF-Tu bound to ribosomes to interact in a productive way with free GTP appears to be a primary reason for inhibition of the turnover of GTPase activity in this condition. In the absence of poly(U), ribosomes are very active in stimulating the kirromycin-induced EF-Tu GTPase either with or without Phe-tRNAPhe, which increases their affinity for the reaction. When Phe-tRNAPhe is present, kirromycin enables either ribosomal subunit to stimulate GTP hydrolysis of EF-Tu, showing that each subunit harbours an active site for the regulation of the catalytic center of EF-Tu. Addition of poly(U) plus Phe-tRNAPhe inhibits the turnover of the kirromycin-induced GTP hydrolysis in the presence of the 30-S subunits, but not of that in the presence of the 50-S subunit, which is inhibited when the 30-S subunit is also added. The 30-S subunit alone appears therefore to be sufficient for formation of a stable complex with EF-Tu and kirromycin. Our results show that expression of EF-Tu GTPase activity coupled with polypeptide synthesis is the result of an highly specific and coordinated mechanism, in which both ribosomal subunits participate and act on the catalytic center of the elongation factor. No kirromycin-like activity was found in the two fragments of the antibiotic molecule, obtained by acid hydrolysis. None of the partial reactions of polypeptide synthesis tested, dependent on elongation factor G, initiation factors or aminoacyl-tRNA-synthetases, appeared to be affected by kirromycin.