Sunday, April 28, 2024
4/28/2024
DESCRIPTION (provided by applicant): The long-term goal of our project has been and is shedding light on the molecular mechanisms involved in protein biosynthesis. Ribosomes, the universal cell organelles facilitating the translation of the genetic code into proteins, are nucleoprotein assemblies (2.3 mDa approximately 4500 RNA nucleotides and approximately 55 proteins) built of two subunits of unequal size, which associate upon the initiation of protein biosynthesis. The immediate objectives of this proposal are: (a) elucidating the detailed mechanisms involved in peptide bond formation, amino acid polymerization, peptide bond formation, translocation, tRNA release, nascent protein progression, elongation arrest and initial steps towards folding. For these studies carefully designed complexes capturing ribosomal particles at defined functional states, are being prepared, based on the high- resolution structures of the two eubacterial ribosomal subunits determined by us. Examples are complexes of substrate analogs, functional ligands, inhibitors, and non-ribosomal compounds relevant to protein biosynthesis, such as native and mutated trigger factor; (b) Determining the parameters acquiring the effectiveness of ribosomal antibiotics, by careful analysis of the distinction between mere binding and inhibitory action, based on antibiotics binding modes to ribosomes of authentic pathogens, of eubacteria serving as pathogen models, and of archaea resembling eukaryotes; (c) Further advance towards understanding mechanisms attaining resistance to ribosomal antibiotics, by elucidating the structural bases of resistance mechanisms developed against traditional as well as advanced ribosomal antibiotics. Methods: High resolution X-ray diffraction data are being collected at cryogenic temperatures from flash- frozen crystals of complexes of functionally active ribosomes, using high brilliance synchrotron radiation. Phases are being determined by a MIRAS, molecular replacement and crystal averaging. The resulting electron density maps are being interpreted interactively and the positions and orientations of the ligands and/or antibiotics are being compared to available biochemical and mutagenesis data. The significance of ribosomal crystallography stems from its potential to illuminate the mechanism of a fundamental life process, protein biosynthesis, as well as to reveal modes of action of antibiotics targeting ribosomes. These studies have already illuminated principles of drug selectivity, and provided significant basic knowledge of possible pathways of drug resistance, hence paving the way to improve therapeutic properties.
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