Targeting the A site RNA of the Escherichia coli ribosomal 30S subunit by 2′-O-methyl oligoribonucleotides:: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics

The bacterial ribosome comprises 30 S and 50 S ribonucleoprotein subunits, contains a number of binding sites for known antibiotics and is an attractive target for selection of novel antibacterial agents. On the 30 S subunit, for example, the A site (aminoacyl site) close to the 3'-end of 16 S rRNA is highly important in the decoding process. Binding by some amino-glycoside antibiotics to the A site leads to erroneous protein synthesis and is lethal for bacteria. We targeted the A site on purified 30 S ribosomal subunits from Escherichia coli with a set of overlapping, complementary OMe (2'-O-methyl) 10-mer oligoribonurcleotides. An equilibrium dialysis technique was applied to measure dissociation constants of these oligonucleofides. We show that there is a single high-affinity region, spanning from A(1493) to C-1510 (K-d, 29-130 nM), flanked by two lower-affinity regions, within a span from U-1485 to G(1516) (K-d, 310-4300 nM). Unexpectedly, addition of the aminoglycoside antibiotic paromomycin (but not hygromycin B) caused a dose-dependent increase of up to 7.5-fold in the binding of the highest affinity 10-mer 1493 to 30 S subunits. Oligonucleotides containing residues complementary to A(1412) and/or A(1493) showed particularly marked stimulation of binding by paromomycin. The results are consistent with high-resolution structures of antibiotic binding to the A site and with greater accessibility of residues of A(1492) and A(1493) upon paromomycin binding. 10-mer 1493 binding is thus a probe of the confonnational switch to the 'closed' conformation triggered by paromomycin that is implicated in the discrimination by 30 S subunits of cognate from non-cognate tRNA and the translational misreading caused by paromomycin. Finally, we show that OMe oligonucleotides targeted to the A site are moderately good inhibitors of in vitro translation and that there is a limited correlation of inhibition activity with binding strength to the A site.