The long term goal of this project is to shed light on the mechanism of protein biosynthesis. Ribosomes are the universal cell organelles facilitating the translation of the genetic code into polypeptide chains. These nucleoprotein assemblies (mw 2.3 mD, about 4500 RNA nucleotides and up to 73 proteins). are build of two subunits of unequal size (0.85 and 1.45 mD) which associate upon the initiation of protein biosynthesis. The immediate objectives of this proposal are to elucidate the molecular structure of the small ribosomal subunit at its activation state and to advance towards the determination of the structure of the large one. Crystals diffracting to about 3 angstrom resolution were grown from both subunits. The methods used for their analysis include crystallography, electron microscopy, biochemistry, metalo-organo chemistry and molecular genetics.. X-ray data are being collected with synchrotron radiation at cryogenic temperatures from flash-frozen crystals and phases are being determined by a combination of MIR, SIRAS and MAD with molecular replacement, exploiting models of ribosomal particles obtained by electron microscopy angular reconstruction. Despite the decay caused by bright synchrotron beam, necessary for collecting the higher resolution data, reliable phases were determined for the small subunit. The 7 angstrom map shows long continuous regions, traced as rRNA, as well as features which can be interpreted as ribosomal proteins. The higher radiation sensitivity of the large subunit crystals, which is accompanied by a low level of isomorphism and instability of cell dimensions, led to 9.5 angstroms MIR map, which may indicate the reasons for the problematic nature of these crystals. The significance of ribosomal crystallography stems from its potential to illuminate one of the fundamental processes of life, protein biosynthesis. Opening not only the possibility to understand pathological deviations of this universal process but also providing the basic knowledge and principles for the design of new therapeutic agents. An important group of proven aminoglycoside antibiotics, such as streptomycin, kanamycin etc., cause misreading of mRNA codons by interacting with the small ribosomal subunit. Hence, detailed structural information should lead to a new generation of more powerful specific and efficient antibiotics.
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