High Resolution Analysis of Ribosome-Bound Nascent Polypeptides by NMR

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DESCRIPTION (provided by applicant): The overall objective of this research is to perform high resolution NMR structural studies of ribosome-nascent chain (RNC) complexes by taking advantage of recently developed NMR pulse sequences for highly sensitive data collection and newly available procedures for the preparation of stable ribosome-bound nascent polypeptides in solution. Several antibiotics acting on the bacterial translation machinery, including erythromycin and telithromycin, act by interfering with the nascent chain progression through the ribosomal exit tunnel. Yet, the lack of high resolution structural characterization of nascent chain-ribosome complexes has prevented the urgently needed progress in understanding the three-dimensional environment upon which these antibiotics exert their action. This lack of atomic resolution information has slowed down rational approaches to the design of novel therapeutics and hampered a deep understanding of the structural role of RNCs within the translational machinery. Based on the working hypothesis that RNCs experience a competition between the tendency to experience intramolecular hydrophobic collapse and the tendency to bind molecular chaperones during translation, the following specific aims will be pursued: (1) NMR structural characterization of ribosome-bound nascent chains (RNCs) of increasing length derived from the natively unfolded protein PIR. (2) NMR structural characterization of RNCs derived from the apomyoglobin sequence in the absence and presence of selected cotranslationally active molecular chaperones. The proposed investigations will be carried out in parallel with purified eubacterial ribosomes from the Escherichia coli and Deinococcus radiodurans pathogens. Preliminary results obtained in the P.I.'s laboratory support feasibility and set the ground for future work. One of the main goals of the National Institute of Allergy and Infectious Diseases is to support basic and applied research to better understand, treat, and ultimately prevent disorders caused by bacterial pathogens. This research project embraces the objectives of this Institute by providing high resolution structural information that will enable and foster the future rational design of therapeutic strategies against infections by E. coli and D. radiodurans. Project Narrative This research project targets NMR structural studies on ribosome-peptidyl-tRNA complexes from Escherichia coli and Deinococcus radiodurans. This research is intended is a pilot study to (a) generate structural data required to gain precious atomic resolution information on ribosome-bound nascent proteins, and (b) to generate high quality structural insights that will aid the future rational design of novel antibiotics. Given that some Escherichia coli strains such as O157:H7 are responsible for serious and sometimes even lethal human disease [e.g., the Summer/Fall 2006 E. coli outbreak in the US, leading to several deaths], the proposed research is directly relevant to human health. Deinococcus radiodurans is the world's most resistant bacterium to UV radiation and, as such, a potentially harmful vector that could be employed for the development of pathogenic strains, possibly to be used as warfare agents. Hence, there are clear connections between the proposed work and human health.