Studies on the Structure and Function of Ribosomes by Combined Use of Chemical Probing and X-Ray Crystallography

The study of the process of protein synthesis, which encompasses translation of the genetic code, has been intensively under way for about 4 decades. This chapter summarizes recent studies of the structure and function of ribosomes by a combination of biochemical and X-ray-crystallographic approaches, focusing primarily on work from the laboratory. Ribosomologists have traditionally used a wide variety of experimental strategies to study the many molecular interactions of the translational machinery. EF-G was bound in the presence of fusidic acid and GTP to ribosomes containing mRNA and tRNA bound to the P site, and hydroxyl radicals were initiated. Three-dimensional crystals of ribosomes and ribosomal subunits were obtained more than a decade ago, but only recently has X-ray crystallography begun to provide detailed information about the large-scale structure of the ribosome. In the X-ray map, bridges from elements originating in the 50S subunit can be seen to contact the minor groove of this helix at positions corresponding closely to those predicted from the foot printing studies. The arrangement of the S8-16S rRNA interaction has been investigated extensively by directed hydroxyl radical probing. Modification interference studies with kethoxal showed that modification of a set of guanines in 16S rRNA interferes with subunit association. The RNA chemical-probing studies left unaddressed the possibility that protein-RNA (or protein-protein) interactions might play a role in subunit association.