CHARACTERIZATION OF THE STRUCTURE AND DYNAMICS OF 16S RRNA A-SITE MUTANTS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 16S ribosomal A-site is directly involved in maintaining the fidelity of mRNA translation by the ribosome. The A-site is also the target of neamine-class aminoglycoside antibiotics. Binding of these antibiotics to the A-site reduces the level of fidelity in translation, resulting in non-functional protein products, resulting in cell death. Recent X-ray crystallographic evidence suggests the positions and contacts of nucleotides A1492 and A1493, within the A-site internal loop, play an important role in maintaining fidelity of mRNA translation. It has been shown that antibiotic binding to the A-site internal loop can distort its structure. The identity of position 1408, opposite to A1492 and A1493 within the A-site internal loop, largely controls the antibiotic binding specificity between Eukaryotes G1408 and Prokaryotes A1408, which is the main rationale behind the clinical success of these antibiotics in treating bacterial infections. In recent years a number of bacterial strains have developed resistance mechanisms to the neamine-class of antibiotics. One mechanism involves mutations in the sequence of the A-site target. Our hypothesis is that mutations of the 16S Ribosomal A-site must maintain a characteristic electrostatic surface for cognate recognition of the mRNA codon by the anti-codon loop of tRNA during translation. To test this hypothesis we are performing NMR studies to solve the solution-state structure for the prokaryotic and eukaryotic wild type A-site, along with several bacterial functional mutants generated in the lab of Dr. Phil Cunningham using his instant evolution methods. This work focuses on the sequence dependence of the A-site conformation and dynamics of positions A1492 and A1493. Binding studies and structure determination of a novel antibiotic, synthesized in the lab of Dr. Mark Spaller, bound to selected constructs are also being performed to explore new avenues in drug design. Comparing these structures to currently published data (NMR, x-ray, footprinting, etc.) will reveal structural and mechanistic aspects of drug binding, as well as provide rationalization of antibiotic resistance and mutant function.