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 of naturally occurring 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 for tRNA translocation, 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 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.

During the course of this research project, we have applied a recently developed experimental device (which we named the `gapped-cp-reconstitution`) that allows to introduce site-specifically non-natural RNA analogues into the catalytic heart of the large ribosomal subunit. Biochemical and recent crystallographic studies revealed the active site of the large ribosomal subunit, the peptidyl transferase center, to be entirely composed of 23S ribosomal RNA. Consequently, catalysis of the two chemical reactions promoted by the ribosome, peptide bond formation and peptide release, are based on a ribozyme mechanism. As ribosomes are so fundamental to life and represent one of the main targets for antibiotics, comprehending how they work is at the heart of molecular understanding of biology. Previous mutational studies of all potential catalytic nucleotides in the peptidyl transferase center turned out to be insufficient to fully understand the chemical mechanisms of protein synthesis. Obviously, the chemical repertoire of different chemical groups that can be placed by introducing natural mutations at active site residues, was too small to unequivocally comprehend peptide bond formation or peptide release at the molecular level. The novel experimental tool that we have set up, however, allows to manipulate single functional groups or even atoms within a ribozyme of up to 1.8 MD size. Our studies revealed a single 2`-hydroxyl group at the ribose sugar at position A2451 of 23S rRNA to be pivotal for catalyzing peptide bond formation. In contrast, we showed that an intact ribose sugar at the 23S rRNA residue A2602 is crucial for efficient peptide release, while having no apparent functional relevance for transpeptidation. These findings underscore the exceptional functional importance of the ribose moiety at A2602 for triggering peptide release. This indicates the catalytic mechanism for peptide bond formation to be distinct from that of peptide release. It is of note that for both ribosome catalyzed reaction, the ribosome provides a 23S rRNA backbone group, rather than any group on a nucleobase despite the universal base conservation.