Reaction mechanisms and structural dynamics of the ribosome

The ribosome is an essential multifunctional particle responsible for protein synthesis in all living cells. The ribosome is composed of two subunits that consist of ribosomal RNA (rRNA) and ribosomal proteins. Decades of biochemical and recent crystallographic studies revealed all ribosomal functions to be rRNA based. Despite the structural insights into the ribosome, the mechanisms of the two main reactions of the large ribosomal subunit, namely peptide bond formation and peptide release, are far from being understood in molecular terms. The aim of the proposed research is to site-specifically introduce modified RNA nucleotides at the active site of the large ribosomal subunit and to unravel the molecular mechanisms of fundamental ribosomal reactions. Since the ribosome is the main target for clinical relevant antibiotics, the knowledge of its functioning is of potential importance for understanding drug resistance and for the future design of new anti-microbial compounds. Because of the complexity of the ribosome and the large size of its RNA components, the site-specific incorporation of a single non-natural nucleotide analog into ribosomal RNA was not possible. However, with the development of a new in vitro reconstitution strategy of the large ribosomal subunit, this problem can be overcome. The idea is to construct a ribosomal RNA whose natural ends are covalently connected and new ends have been introduced elsewhere in the molecule (circularly permutated rRNA). These new endpoints will be chosen to be located in close proximity to the active site and serve to site-specifically position a modified nucleotide within the ribosome. Subsequently, this modified ribosomal RNA will be used together with the ribosomal proteins to assemble large subunits. It is then possible to study the functional effects of this unique nucleotide modification on peptide bond formation and peptide release. Additionally, this technique is also applicable to study ribosomal dynamics during protein synthesis. In this case a photo-reactive nucleotide will be incorporated at the new endpoint of the circularly permutated rRNA. Photo-crosslinking studies of ribosomes arrested in different functional states can reveal conformational changes of the ribosome during protein synthesis.