THE ANTIBIOTICS MICROCOCCIN AND THIOSTREPTON INTERACT DIRECTLY WITH 23S RIBOSOMAL-RNA NUCLEOTIDES 1067A AND 1095A

The antibiotics thiostrepton and micrococcin bind to the GTPase region in domain II of 23S rRNA, and inhibit ribosomal A-site associated rea ctions. When bound to the ribosome, these antibiotics alter the access ibility of nucleotides 1067A and 1095A towards chemical reagents. Plas mid-coded Escherichia coil 23S rRNAs with single mutations at position s 1067 or 1095 were expressed in vivo. Mutant ribosomes are functional in protein synthesis, although those with transversion mutations func tion less effectively. Antibiotics were bound under conditions where w ild-type and mutant ribosomes compete in the same reaction for drug mo lecules; binding was analysed by allele-specific footprinting. Transve rsion mutations at 1067 reduce thiostrepton binding more than 1000-fol d. The 1067G substitution gives a more modest decrease in thiostrepton binding. The changes at 1095 slightly, but significantly, lower the a ffinity of ribosomes for thiostrepton, again with the G mutation havin g the smallest effect. Micrococcin binding to ribosomes is reduced to a far greater extent than thiostrepton by all the 1067 and 1095 mutati ons. Extrapolating these results to growing cells, mutation of nucleot ide 1067A confers resistance towards micrococcin and thiostrepton, whi le substitutions at 1095A confer micrococcin resistance, and increase tolerance towards thiostrepton. These data support an rRNA tertiary st ructure model in which 1067A and 1095A lie in close proximity, and are key components in the drug binding site. None of the mutations alters either the higher order rRNA structure or the binding of r-proteins. We therefore conclude that thiostrepton and micrococcin interact direc tly with 1067A and 1095A.