Translocation mechanism of ribosomes

The paper summarizes studies of the molecular mechanism of the dynamic function of the ribosome, i. e. translocation, performed in the author's laboratory during the past decade. The hypothesis of the locking-unlocking of the ribosomal subparticles and the kinematical model of the working ribosome, the processes of spontaneous (factor-free) and factor-dependent translocation, the sequence of events in the factor-dependent translocation, the energetics of translocation and the contribution of the elongation factors with GTP are considered. The following conclusions are made: (1) the translocation mechanism is intrinsic to the structural organization of the ribosome itself but not introduced by the protein elongation factors; (2) the transpeptidation reaction is one of the sources of energy for the work of the translocation mechanism; (3) the protein elongation factors with GTP impart additional energy to the ribosome, including that for translocation, and thus ensure excess power which is realized, in particular, in the increase of the translocation rate and its resistance against inhibitors and hindrances; (4) the promoting role of the elongation factors with GTP does not proceed by a direct conjugation of GTP hydrolysis with translocation, but through the affinity of the elongation factors to the ribosome, with a subsequent compensation of the affinity at the expense of GTP cleavage.     

Interaction of tRNA with ribosomes

Definition of the site of tRNA-binding to ribosomes is suggested on the basis of a free energy of tRNA-ribosome interaction. From this point of view disagreements that have arisen in recent years concerning the numbers of tRNA binding sites on the ribosome, their distribution between subunits, the properties of the third site E in ribosomes and the compatibility of new experimental data with different models of elongation cycle are discussed. The observation of the third site in the ribosome (messenger independent and with a presumably exit function) is not a refutation but an extension of Watson's model of translating ribosome.     

Structure and evolution of ribosomes

Ribosomes are the only cell organelles occurring in all organisms. E. coli ribosomes, which are the best characterized particles, consist of three RNAs and 53 proteins. All components have been isolated and characterized by chemical, physical and immunological methods. The primary structures of the RNAs and of all the proteins are known. Information about the secondary structure of the proteins derives from circular dichroism measurements and from secondary structure prediction methods. The tertiary structure is being studied by limited proteolysis, proton magnetic resonance and crystallization followed by X-ray analysis. Various methods are being used to elucidate the architecture of the ribosomal particle: three-dimensional image reconstruction of crystals of bacterial ribosomes and/or their subunits; immune electron microscopy; neutron scattering; protein-protein, protein-RNA and RNA-RNA crosslinking; total reconstitution of ribosomal subunits. The results from these studies yield valuable information on the architecture of the ribosomal particle. Many mutants have been isolated in which one or a few ribosomal proteins are altered or even deleted. The genetic and biochemical characterization of these mutants allows conclusions about the importance of these proteins for the function of the ribosome. Ribosomal proteins from various prokaryotic and eukaryotic species have been compared by two-dimensional gel electrophoresis, immunological methods, reconstitution and amino acid sequence analysis. These studies show a strong homology among prokaryotic ribosomal proteins but only a weak homology between proteins from prokaryotic and eukaryotic ribosomes. Comparison of the primary and secondary structures of the ribosomal RNAs from various organisms shows that the secondary structure of the RNA molecules has been strongly conserved throughout evolution.