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.
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.
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Arthur
A
Abelian
Andrew P
AP
Walsh
Georg
G
Lentzen
Fareed
F
Aboul-Ela
Michael J
MJ
Gait
D000617:Aminoglycosides / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D003956:Dialysis / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D008958:Models, Molecular; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects*; D021122:Protein Subunits / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D009843:Oligoribonucleotides; D021122:Protein Subunits; D012335:RNA, Ribosomal