Structural studies of the ribosome: rRNA building blocks characterization and interactions analysis

The first high resolution crystal structures initiated a new era in ribosomal studies in which investigations depend on knowledge of atomic coordinates. The studies I conducted on the three-dimensional structure of the ribosome aim to reveal and understand the ribosome modular organization, and at the same time to investigate protein-RNA interaction mechasisms. All the results presented in this thesis refer to the case study of the large ribosomal subunit 50S of Haloarcula marismortui, omitting the 5S rRNA. Three-dimensional RNA motifs are the basic building blocks of ribosomal RNA (rRNA) architecture. A novel method to search for these substructures, based on shape histograms, is presented in Chapter 2. The shape histogram is a vector representation of the distribution of distances between a set of points and a fixed one. Shape histograms are not only an efficient tool to compare RNA fragments, but also they well capture the structural similarities behind the flexible and highly variable structure of ribosomal RNA. The method outperforms existing ones in time efficiency, reaching the same level of correctness. Branching points of three or more helices, named junctions, are the most variable and complex loops of rRNA, and the least characterized. Shape histograms together with angles between inter-helical fragments provide major informations on the 3D conformation of junctions: angles give informations on the eccentricity of the loop, while shape histogram range and distribution reveal the folding. Based on these features I propose a possible classification of these motifs with respect to their 3D conformation. Finally in Chapter 4 I dissect protein-RNA interactions within the ribosome. Statistical analysis reveal distinguishing features of the ribosome with respect to protein interactions with other RNA molecules, such as the dominant role of the ribose group. Furthermore protein interaction mechanisms with known RNA motifs are investigated, in particular with standard tetraloops, kink-turns, and single extruded nucleotides in general. A consensus interaction pattern is detected for protein contact surfaces with standard tetraloops, characterized by dense areas of contacts made by positive residues (mainly arginine). Ribosomal proteins also reveal a characteristic binding site with kink-turns in correspondence of the extruded base. Due to its shape, this site has been called tripod. Tripods proved to be common to several single extruded nucleotides. Besides a conserved shape, tripods show a preference towards purines, especially adenine, and typically make hydrogen bonds.