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Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA
D Fourmy1, S Yoshizawa, J D Puglisi
1Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 95064, USA.
Abstract:
Aminoglycoside antibiotics that bind to ribosomal RNA in the aminoacyl-tRNA site (A-site) cause misreading of the genetic code and inhibit translocation. An A-site RNA oligonucleotide specifically binds to aminoglycoside antibiotics and the structure of the RNA-paromomycin complex was previously determined by nuclear magnetic resonance (NMR) spectroscopy. Here, the A-site RNA structure in its free form has been determined using heteronuclear NMR and compared to the structure of the paromomycin-RNA complex. As in the complex with paromomycin, the asymmetric internal loop is closed by a Watson-Crick base-pair (C1407.G1494) and by two non-canonical base-pairs (U1406.U1495, A1408.A1493). A1492 stacks below A1493 and is intercalated between the upper and lower stems. The comparison of the free and bound conformations of the RNA shows that two universally conserved residues of the A site of 16 S rRNA, A1492 and A1493, are displaced towards the minor groove of the RNA helix in presence of antibiotic. These changes in the RNA conformation place the N1 positions of A1492 and A1493 on the minor groove side of the A-site RNA and suggest a mechanism of action of aminoglycosides on translation.
Insights
Aminoglycoside antibiotics disrupt bacterial translation by binding to ribosomal RNA. This study reveals how antibiotic binding alters RNA structure, affecting key residues and suggesting a mechanism for their action.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Aminoglycoside antibiotics target the ribosomal A-site RNA, causing genetic code misreading and inhibiting protein synthesis.
- Previous studies determined the structure of the RNA-paromomycin complex using NMR spectroscopy.
Purpose of the Study:
- To determine the free form structure of the A-site RNA using heteronuclear NMR.
- To compare the free RNA structure with the paromomycin-bound structure.
- To elucidate the conformational changes induced by aminoglycoside binding.
Main Methods:
- Heteronuclear Nuclear Magnetic Resonance (NMR) spectroscopy was employed to determine the structure of the free A-site RNA.
- Comparative structural analysis was performed between the free and paromomycin-bound RNA forms.
Main Results:
- The free A-site RNA structure was determined, revealing specific base-pairing interactions.
- Comparison with the paromomycin-bound structure showed that conserved residues A1492 and A1493 are displaced towards the minor groove upon antibiotic binding.
- These conformational changes position key nitrogen atoms (N1) of A1492 and A1493 on the minor groove side.
Conclusions:
- The study reveals significant conformational changes in the A-site RNA upon aminoglycoside binding.
- The displacement of A1492 and A1493 and their altered positioning suggest a direct role in the mechanism of aminoglycoside action.
- These findings provide insights into how aminoglycosides interfere with bacterial translation.