X-ray crystallographic studies of the large ribosomal subunit

The large ribosomal subunit is responsible for peptide bond formation during protein synthesis. In this study, structures of the large ribosomal subunit of the halophilic achaeon Haloarcula marismortui , determined by X-ray crystallography, are used to investigate RNA structure and ribosomal function.; Analysis of the structure of Haloarcula marismortui large ribosomal subunit has revealed a common RNA structure that we call the K-turn. This helix-internal loop-helix motif is characterized by a kink in the phosphodiester backbone and a sharp turn in the RNA helix, and is often recognized by proteins. Six K-turns are identified in H. marismortui 23S rRNA and four in previously solved RNA structures. A consensus sequence for the K-turn predicts its widespread presence in nature.; Structures of the large ribosomal subunit in complexes that represent states before, during and after the peptidyl transferase reaction have been determined. Combining the complex of a P-site substrate with the separate A-site substrate complex gives a model of the peptidyl transferase center directly before reaction. The alpha-amino group is positioned to attack the carbonyl carbon of P-site substrate by hydrogen bonding to N3 of A2486 (E. coli 2451). Complexes with new transition state analogues and a computationally generated transition state suggest that the oxyanion of the transition state of the peptidyl transferase reaction interacts with a solvent molecule near U2620 (2585), not N3 of A2486 (2451). The structure obtained when substrates are diffused into large subunit crystals shows the formation of products. This result, along with biochemical experiments, shows the crystalline ribosomes to be catalytically active. The resulting structure represents a state that occurs after peptide bond formation, but before the hybrid state of protein synthesis.; The ribosomal E-site binds deacylated-tRNA. The structures of complexes between the H. marismortui 50S subunit and two different E-site substrates have been determined. The E-site substrates bind in the center of the loop-extension of protein L44e, and make specific contacts with both L44e and 23S rRNA. These structures are consistent with data that identified the E-site biochemically, and explain the specificity of the E-site for deacylated tRNAs, as the site is too small for any relevant aminoacyl-tRNA.