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Title: NMR structural studies of the binding of peptidyl transferase antibiotics to conserved secondary structural motifs of 23S ribosomal RNA
Author: Nareen, Misbah
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2011
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Abstract:
The peptidyl transferase centre (PTC) of 23S ribosomal RNA is the target for a number of antibiotics which inhibit protein synthesis. The precise mode of binding of these antibiotics is largely unknown and hence is an active area of research in structural biology. The NMR solution structures of three PT antibiotics, bamicetin, sparsomycin and anisomycin have been successfully characterised using a range of two-dimensional NMR techniques and restrained molecular dynamics. The NMR structures of the these antibiotics provided valuable first hand insight into their conformations, since no X-ray crystal structures of the antibiotics in their free states have been determined so far. Bamicetin adopts a folded conformation possibly held by intramolecular hydrogen bonds and similar to the published NMR structure of amicetin. These antibiotics generate spontaneous single nucleotide mutants upon prolonged exposure and bamicetin and sparsomycin are universal PT inhibitors, interacting with all three evolutionary domains of 23S rRNAs. The amicetin antibiotic produces a spontaneous single mutation U2457C in the Halobacterium halobium (H.hal) 23S rRNA and the binding site is predicted to be very close to this nucleotide. The similarity in chemical structure with amicetin, suggests bamicetin to target the same binding site on the 23S rRNA. Both bamicetin and sparsomycin show exchange retarded amide proton resonances in the NMR spectrum, akin to other amicetin family antibiotics, indicating the retarded exchange to be a characteristic feature in the native solution state. The Bacillus subtilis (B.subtilis) 70S ribosomes have strong affinity for bamicetin and so a highly conserved 27mer RNA motif containing the possible binding site was selected for NMR structure determination and bamicetin binding studies. The greater number of imino proton resonances observed together with the high quality of the determined structure of the motif proved that B.subtilis rRNA is more stable than E.coli and H.hal rRNAs. The B.subtilis 27mer rRNA-bamicetin interaction studies revealed a fast exchange, weak binding system and careful analysis of line width and chemical shifts indicated changes at the local conformation of the RNA after binding. To probe the cross-hypersensitivity phenomenon, a 25mer RNA corresponding to the thiostrepton-resistant mutant (G1159) residing in the domain II of H.hal 23S rRNA was chosen for NMR structure determination and amicetin binding. Discrete chemical shift changes and NOESY experiments using ultrahigh field 1GHz NMR revealed weak interactions. The structures of the antibiotics and analysis of their dynamics as well as interactions with the RNA motifs of different organisms have yielded important information in understanding their binding and inhibitory activities at the atomic level. The results can be used for generating new or hybrid antibiotics to tackle the escalating problem of antibiotic resistance.
Supervisor: Ramesh, Vasudevan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.553299  DOI: Not available
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