Evolution and assembly of the ribosome

The ribosome is a large macromolecular assembly responsible for protein synthesis in all living cells. A typical bacterial ribosome consists of three ribosomal RNA (rRNA) molecules and approximately fifty ribosomal proteins (r-proteins), which are arranged into two subunits of unequal size and distinct function. The large subunit promotes formation of the peptide bond, and the small subunit enforces the recognition between the mRNA codons and the tRNA anticodons. With the availability of the ribosome crystal structure, it becomes clear that the two major functions, peptide bond formation and decoding, are performed within an entire RNA environment devoid of proteins. Combining with the fact that the majority of the ribosomal components are conserved across all three domains of life, it is believed that the ribosome has its origin deep in the RNA world before the last universal common ancestor (LUCA). Further evolutionary studies lead to the hypothesis that the evolution of the ribosome begins with a prototype ribozyme that catalyzes peptidyl-transferase reaction. Structural and sequence analysis suggests that the small ribozyme capable of catalyzing formation of short peptides may still exist in the core of the modern ribosome. Accordingly, a proto-ribosome model is constructed computationally using RNA fragments near the peptidyl-transferase center (PTC), and is proven to be stable throughout the micro-second molecular dynamics (MD) simulations. The model is capable of incorporating freely diffusing substrates spontaneously into its binding site, and holds them in both pockets long enough to reach a transition intermediate favorable for peptide bond formation. This in silico designed proto-ribosome is then subjected to experimental investigations to test its ability to assemble and bind potential substrates in solution. The successful design of the proto-ribosome presents a possible scenario for the initial development of the early translation apparatus. The proto-ribosome coupled with the probable parallel evolution of ancient tRNAs might have driven the emergence of the oldest coded protein shortly afterwards. The universally conserved r-protein S4 is likely an ancient protein due to its role in the initiation of the 30S assembly, control of the translational accuracy, and regulation of the conserved operon. However, the N-terminal domain of S4 is identified as a “molecular signature” that distinguishes between Bacteria and Archaea, and hence might be a newer addition to the protein. The presence of both an old and a new component in the same protein makes it an extremely interesting case to study for the ribosomal evolution. Therefore, we perform phylogenetic analysis of S4 in relation to a broad sharing of zinc/non-zinc binding sequence in the N-terminal domain of the protein, and study the scope of horizontal gene transfer (HGT) of S4 during bacterial evolution. The complex history presented for “core” protein S4 suggests the existence of a gene pool before the emergence of bacterial lineages and reflects the pervasive nature of HGT in subsequent bacterial evolution. We then continue the study to understand the molecular driving force for such differentiated evolutionary history concerning one single protein. Consistency between experimental measurements and all-atom MD simulations indicates that the addition of the disordered N-terminal domain of S4 coevolved with a molecular signature in the rRNA helix h16, to couple the folding and binding process and accelerate the protein:RNA recognition. The coupling between protein and RNA molecules in both evolution and modern dynamics inspired further study of the protein:RNA interactions during early assembly of the bacterial 30S small subunit. A practical protocol of all-atom MD simulation combined with RNA conformation clustering is developed to probe the folding landscape of the RNA molecules. In addition, the structure-based G¯o-potential is developed within the framework of the all-atom molecular dynamics CHARMM force field, with which hundreds of simultaneous folding and binding events between the rRNA and r-protein are captured. Comparison between these simulations with the smFRET experiments reveals folding pathways constructed upon distinct structural intermediates of the RNA molecule. Our studies illustrate the complex nature of RNA folding in the presence of a protein binding partner, and provide insight into the role of population shift and the induced fit in the protein:RNA recognition process. The methodology developed therein will facilitate future studies of the ribosomal assembly on increasingly larger scale