Crystallographic, Biochemical and Genetic Studies on Halophilic Ribosomes

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Summary Ribosomal particles from Haloarcula marismortui were crystallized. The best crystals, diffracting to 2.9 A resolution and yielding crystallographic data of reasonable quality were grown from the large ribosomal subunits. Attempts at crystallization of functional ribosomal complexes are in progress, benefiting from experience gained from crystals of ribosomes of an extreme thermophilic bacterium, Thermus thermophilus . For obtaining phase information, a monofunctional reagent was prepared from an undecagold cluster, by attaching to it a chemically reactive handle, specific for sulfhydryl moieties. Heavy atom derivatives were prepared by binding this cluster to exposed sulfhydryls prior to the crystallization. Cores of halophilic ribosomal particles, lacking four ribosomal proteins, were prepared using dioxane. All detached proteins could be fully reconstituted. However, blocking the -SH group of one of them (HmaL11), prevented its incorporation into the core particles. The so obtained depleted 50S subunits crystallize under the same conditions as native ones and show apparent isomorphism with them. Most of the genes of several r-proteins were cloned. These are being used for sequencing as well as for providing new locations for binding heavy atom clusters by genetic insertion of cysteines on the ribosomal surface, according to accessibility data, obtained either chemically or by limited proteolysis. A nucleoprotein complex of protein HmaL1 and a fragment of the 23S rRNA was isolated from ribosomes. Chimeric complexes were reconstituted with E. coli ribosomal components, indicating rather high homolgy, despite the evolution distance.