Inhibition of Antiassociation Activity of Translation Initiation Factor 3 by Paromomycin

Abstract

The effect of paromomycin on the interaction of ribosomal subunits was studied. Paromomycin inhibited the antiassociation activity of initiation factor 3 (IF3). Furthermore, ribosomal subunits were associated to form 70S ribosomes by paromomycin even in the presence of 1 mM Mg 2؉ . Paromomycin did not inhibit the binding of IF3 to the 30S ribosomal subunits. On the other hand, IF3 bound to the 30S subunits was expelled by paromomycin-induced subunit association (70S formation). These results indicate that the stabilization of 70S ribosomes by paromomycin may in part be responsible for its inhibitory effects on translocation and ribosome recycling.Aminoglycosides cause miscoding (6, 18), stabilization of 70S ribosomes (4, 26), inhibition of translocation (2,10,16,24), alanylation of transfer mRNA (37), and recycling of ribosomes (12,13,17). Although the mechanisms of the miscoding effect of paromomycin have been worked out previously (27)(28)(29), the exact mechanism of the effect of paromomycin on the translocation and recycling steps remains obscure. We investigated the effect of paromomycin on the interaction of subunits which plays an important role in both of these steps. We show that paromomycin inhibits the antiassociation activity of initiation factor 3 (IF3), which is an important component of the disassembly reaction of posttermination ribosomal complexes (14). This inhibitory effect comes from the fact that paromomycin strengthens the interaction between ribosomal subunits and even induces association of the subunits at low Mg 2ϩ concentrations. Upon the association of subunits by paromomycin, the 30S-bound IF3 is displaced from the ribosomes. A possible mechanism of the inhibitory action of paromomycin on the translocation step is discussed. MATERIALS AND METHODSBuffers. Buffer J consisted of 10 mM Tris-HCl, pH 7.4, 10 mM MgSO 4 , 50 mM KCl, and 1 mM dithiothreitol (DTT). Buffer R consisted of 10 mM Tris-HCl, pH 7.4, 8.2 mM MgSO 4 , 84 mM NH 4 Cl, and 0.5 mM DTT. Buffer S consisted of 10 mM Tris-HCl, pH 7.4, 1 mM MgSO 4 , 84 mM NH 4 Cl, and 0.5 mM DTT. Buffer S2 consisted of 10 mM Tris-HCl, pH 7.4, 4 mM MgSO 4 , 84 mM NH 4 Cl, and 0.5 mM DTT. Buffer S3 consisted of 10 mM Tris-HCl, pH 7.4, 6 mM MgSO 4 , 84 mM NH 4 Cl, and 0.5 mM DTT.Ribosomes and factors. Vacant ribosomes were prepared from Escherichia coli MRE600 (purchased from the University of Alabama Fermentation Facility, Birmingham) as described previously (21). Ribosome recycling factor (RRF) and elongation factor G (EF-G) were purified as described previously (11, 21) from E. coli DH5␣ harboring plasmid pRR2 (34) and E. coli JM83 harboring plasmid pECEG (15) (obtained from P. March), respectively. IF3 was purified from E. coli XL1-Blue harboring a plasmid expressing His-IF3 (35) (obtained from T. Ueda).Sucrose density gradient ultracentrifugation (SDGC). Unless otherwise mentioned, 0.6 A 260 units (approximately 14 pmol) of 70S ribosomes (or the dissociated subunits) or 0.45 A 260 units (approximately 31 pmol) of isolated 30S subunits in 275 l...

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