Despite massive studies on protein biosynthesis, a full understanding of this process still awaits a reliable molecular model. The long term goal of this project is to illuminate the molecular mechanism of protein biosynthesis. Ribosomes are the universal cell organelles which facilitate this process. The immediate objective is to elucidate its three-dimensional structure, employing X-ray crystallography. Bacterial ribosomes are assemblies of some 57 different proteins and three RNA chains with no internal symmetry and a total molecular weight of approximately 1.5 million daltons. They are notoriously unstable and flexible, hence very difficult to crystallize. Nevertheless, experimental procedures for reproducible production of suitable crystals from ribosomal particles of thermophilic and halophilic bacteria, for collecting crystallographic data from these crystals and for evaluating the data, have been developed. The X-ray crystallographic studies are being supported by electron microscopy, image reconstruction, neutron diffraction, biochemistry and genetics manipulation. Crystallographic studies, using an intense synchrotron X-ray beam at cryo- temperature, are being carried out on crystals of whole ribosomes, their complexes with components which participate In the process of protein biosynthesis, their natural, mutated and modified large and small subunits are currently being carried out. Of particular interest are complexes of whole ribosomes, at distinct functional states, together with mRNA and charged tRNA, of the large ribosomal subunits with tRNA and a fragment of the nascent protein chain, of mutated particles, missing a ribosomal protein, and of modified particles, whose free -SH groups have been covalently and quantitatively bound to a super-dense heavy-atom cluster (undecagold). A monofunctional reagent of this cluster have been designed for these studies, and preliminary results indicate its suitability for phasing. Comparative studies between free and bound ribosomal subunits should illuminate the conformational changes occurring upon the association of the subunits and the initiation of protein biosynthesis. Well designed heavy atom probes should enable the locations of selected steps In protein biosynthesis. Messenger RNA, coding for the synthetic sequences as well as for naturally occurring proteins, will be used for in vitro biosynthesis The length of the newly synthesized oligopeptide will be controlled by omitting a selected amino acid from the reaction mixture. The extent of binding of the various nascent chains to the ribosomes will be measured. The firmly bound complexes will be crystallized. The significance of these studies stems from the fundamental value of the increased understanding of a basic life process, as well as from its potentials applicative aspect. Thus, elucidation of the effects of antibiotics at the molecular level should become possible once the structure of ribosomes is known, since some of the antibiotics block various steps of protein biosynthesis. Furthermore, results of these studies may provide the basic principles for the design of powerful and efficient therapeutic agents and shed light on abnormal and pathological deviations. Last, but not least, these studies are bound to contribute to the development of sophisticated crystallographic experimental methods.
Showing the most recent 10 out of 83 publications
