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

Abstract

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 aminoglycoside 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 oligoribonucleotides. An equilibrium dialysis technique was applied to measure dissociation constants of these oligonucleotides. We show that there is a single high-affinity region, spanning from A1493 to C1510 (Kd, 29–130 nM), flanked by two lower-affinity regions, within a span from U1485 to G1516 (Kd, 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 A1492 and/or A1493 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 A1492 and A1493 upon paromomycin binding. 10-mer 1493 binding is thus a probe of the conformational 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.

Analysis of ribosomal inter‐subunit sites as targets for complementary oligonucleotides

“…The oligonucleotide B2a‐16S_1, which binds to the decoding A site, bound the 30S subunits and 70S ribosomes with similar K d values of 100 ± 40 and 80 ± 30 nM, respectively, while no binding of B2a‐16S_1 to the 50S ribosomes was observed up to a concentration of 1 µM (Table ). In a previous study of the same oligonucleotide an equilibrium dialysis assay provided a K d value of 29 ± 4 nM for its binding to the 30S subunits, which is comparable to the value obtained here.…”

“…To test the efficiency of designed oligonucleotides in translation inhibition the half‐maximal inhibitory concentrations (IC 50 ) were determined for each of riRNAs (Figure and Supporting Information Table S3). The anti‐SD oligonucleotide, which binds to the anti‐Shine‐Dalgarno region in 16S rRNA, and B2a‐16S_1, which binds to the A site, were used as controls, because of their known efficiency in translation inhibition . Indeed, anti‐SD was the most efficient with an IC 50 of 0.2 ± 0.1 µM, while B2a‐16S_1 had an IC 50 of 0.8 ± 0.5 µM, which confirms its previously reported sequence‐specific inhibitory potential .…”

“…The anti‐SD oligonucleotide, which binds to the anti‐Shine‐Dalgarno region in 16S rRNA, and B2a‐16S_1, which binds to the A site, were used as controls, because of their known efficiency in translation inhibition . Indeed, anti‐SD was the most efficient with an IC 50 of 0.2 ± 0.1 µM, while B2a‐16S_1 had an IC 50 of 0.8 ± 0.5 µM, which confirms its previously reported sequence‐specific inhibitory potential . Out of the 22 tested riRNAs five displayed IC 50 below 1 µM, eight had IC 50 in the range 1 to 1.5 µM, and the remaining ones above 1.5 µM.…”

“…The methyl group in the 2′ position of ribose in 2′‐OMe RNA is expected to enhance its affinity to rRNA in addition to increasing nuclease resistance. It was previously shown that the binding of 2′‐OMe RNAs to the ribosome can inhibit translation …”

Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding

“…Recent elegant experiments (24) coupling X-ray crystallography and fluorescence of 2-amino purine substituted A1492 and A1493 fully confirm that interpretation. A different set of experiments (28), using equilibrium dialysis of 2′-O-methyl oligoribonucleotides devised for binding to the A site of 30S particles, came to a similar conclusion. The observed conformational change is necessary to allow A1492 and A1493 to interact specifically with the first two of the three base pairs formed by the cognate codon–anticodon interaction.…”