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.

During the course of this research project, we have developed and applied a novel 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 that the active site of the large ribosomal subunit, the peptidyl transferase center, is composed entirely of 23S ribosomal RNA. Consequently, catalysis of the two main 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 understand the mechanism how amino acids are connected during protein synthesis. Obviously, the chemical repertoire of different chemical groups that can be placed by introducing natural mutations into active site residues, was too small to unequivocally understand peptide bond formation 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. We have applied the `gapped-cp-reconstitution` to identify potential functional groups at twelve universally conserved active site nucleosides. 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 to previous theories, the adenine base at A2451 does not harbor any critical groups for forging a peptide bond, at least under the in vitro conditions applied. The data explain why the importance of the A2451 2`-hydroxyl escaped the detection by regular mutagenesis studies, since this group remained untouched in all the previous mutants, thereby highlighting the potential of the developed experimental procedure. We have also optimized experimental conditions to apply this molecular tool to study other ribosome promoted reactions such as peptide release during the termination phase of protein biosynthesis as well as tRNA movement during the translocation phase of the ribosomal elongation cycle.