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.

Figure 1. Secondary structure of a section of the 3′-end of 16 S rRNA of E. coli showing the A site and the nucleotide numbers

Residues within the box are targeted by the series of OMe oligonucleotides used in the present study (see Figure 3). Helix numbers (H44 and H45) are marked. Modifications: m⁴Cm, N-4-methyl-2′-O-methylC; m⁵C, 5-methylC; m³U, N-3-methylU; m²G, N-2-methylG; m₂⁶A, N-6-dimethylA.

Figure 2. Filter-binding assay

(A) Effect of the OMe 1487 oligomer length on the binding to 30 S ribosomal subunits. All oligomers have their 3′-ends complementary to residue 1487 in 16 S RNA. Complementary 12-mer (•), 10-mer (▴), 8-mer (▪), 6-mer (♦) and a random 10-mer (×). (B) Binding curves of the OMe 10-mers 1485 (▪), 1489 (○), 1493 (▵), 1495 (▴), 1501 (•), 1505 (⋄), 1507 (♦). For details of oligomers used, see Figure 3. Results are means±S.D. for four replicate experiments.

Figure 3. Scan of binding strength through use of equilibrium dialysis

Across the top: the targeted sequence of the 16 S rRNA. Grey-shaded nucleotides are those not seen to be in hydrogen-bonded pairings according to the crystal structure of the 30 S subunit [29]. From left to right: the code number of the oligomer, its sequence, and its measured K_d (±S.D.; nM) for binding to the 30 S subunit.

Figure 4. Competition with antibiotics

(A) Dose-dependency on the K_d values of the OMe 10-mer 1493. Paromomycin (•), hygromycin B (▪), anisomycin (⋄), chloramphenicol (▵). (B) Effects of 300 μM hygromycin B (grey bars) or paromomycin (white bars) on the K_d values of the 10-mers. Black bars, no antibiotic. Results are means±S.D. for four replicate experiments.

Figure 5. In vitro translation inhibition (black bars; left y-axis) and binding strength (grey bars; right y-axis) of the oligomers

Results are means±S.D. for four replicate experiments.

Figure 6. Binding of antibiotics to the bacterial ribosomal 30 S subunit

(A) Representation of the ribosomal 30 S subunit from Thermus thermophilus when bound by ‘near-cognate’ tRNA analogue and paromomycin {[29]; Protein Data Bank (PDB) code 1N33}, highlighting the residues (except A¹⁴⁹³) targeted for base-pairing complementarity with the OMe 10-mer 1493 (green) and additional residues targeted by 10-mers 1491, 1495, 1497, 1499 and 1501 (yellow). Remaining residues targeted by other oligonucleotides used in the present study are in red. Residues A¹⁴⁹² and A¹⁴⁹³ are shown in liquorice format in light blue. Non-targeted nucleotides are in purple. Proteins are shown in grey. The view is from the subunit interface. (B) Close-up view of the highlighted region shown in (A), with paromomycin shown in white and RNA residues complementary to targeted residues in purple. The view is rotated approx. 180° about the y-axis relative to that shown in (A). Proteins and near-cognate tRNA analogue together with non-targeted regions of ribosomal RNA are not shown. Colour coding is as in (A). (C) Analogous view of the structure of hygromycin B bound to the 30 S subunit ([35]; PDB code 1HNZ). Colour coding is as in (A) and (B).