Nucleotide analog interference in the ribosome

The ribosome is an evolutionarily ancient enzyme that plays a central role in the cell metabolism. The ribosome is a multifunctional ribonucleoprotein (RNP) particle composed of two unequal subunits that translates the genome`s message into all proteins needed for life. Its crucial role in gene expression is mirrored by the fact that the ribosome represents the main target for clinical relevant antibiotics. Therefore the knowledge of its functioning is of potential importance for understanding drug resistance and for the future design of new anti-microbial compounds. Solving the crystallographic structure of the ribosome brought about a quantum leap in the understanding of the ribosome organization. These studies finally revealed the ribosome as an RNA enzyme since all functionally important sites were identified to be composed almost entirely of ribosomal RNA (rRNA). Despite these structural insights, the catalytic mechanisms of the two main reactions promoted by the large ribosomal subunit, namely peptide bond formation and peptide release, as well as the detailed events required to assemble this enormous RNP complex, are far from being understood in molecular terms. The aim of the proposed research is to chemically engineer the active site of the ribosome by applying a recently developed procedure (the `gapped-cp-reconstitution`), that allows to site-specifically introduce a single modified RNA nucleoside at a specific rRNA site of the large ribosomal subunit. This approach significantly enlarges the pool of chemically distinct residues that can be placed specifically into the large ribosomal subunit compared to standard mutagenesis, which is limited to only three possible mutations. This allows the investigation of fundamental ribosomal functions, such as peptide bond formation, peptide release, or translocation with thus far unequalled precision. Additionally, the new `gapped-cp-reconstitution` approach has the potential to deepen our understanding on molecular events driving large ribosomal subunit assembly.

The ribosome is an essential and evolutionarily ancient biocatalyst that plays a central role in all cells. The ribosome, composed primarily of ribosomal RNA, translates the genomes message into all proteins needed for life. In the course of this research project we have developed and employed a novel experimental method allowing us to exchange and manipulate single atoms of the ribosomal RNA within the context of this central enzyme. This allowed the investigation of the different fundamental ribosomal functions during protein biosynthesis with thus far unequalled precision. We were able to pin-point functional groups and atoms within the ribosome that play central roles during the different steps of protein synthesis. The crucial role of the ribosome for biology is mirrored by the fact that the ribosome represents the main target for clinical relevant antibiotics. Therefore the knowledge of its functioning on the molecular level is of utmost importance for understanding drug resistance and for the future design of new antimicrobial compounds.