Final Report Summary - RSDYN (Ribosome dynamics analysed by novel cross-linking/mass spectrometry)
The aim of the project was to study the dynamics of ribosome structure by the use of cross-linking/mass spectrometry. We focussed on the development and use of two different methodologies for the task – protein:RNA and protein:protein crosslinking.
For protein:RNA interactions, we sought to develop a means for the identification of the peptide, and the exact amino acid that is crosslinked to RNA during UV irradiation in living cells. This is based on the identification of the specific mass difference resulting from the single residual nucleotide that remains associated with the peptide following complete hydrolysis of the RNA and partial digestion of the protein. We precisely mapped the RNA-binding sites of hundreds of yeast proteins following crosslinking in actively growing cells, validated by using available high-resolution structures for yeast ribosome and individual protein-RNA complexes, together with genome-wide analysis of RNA targets of the novel RNA-binding protein enolase. This approach should be widely applicable for the reliable and accurate characterization of the protein-RNA interactome in many systems. We are applying it to ribosome biology at the moment and developing it further. Protein:protein crosslinking was applied to study the changes in composition and structure of Escherichia coli ribosomes from ribosome assembly-deficient strains. First results confirmed applicability of the proposed methods for the task, further work is in progress.
The combination of protein:RNA and protein:protein crosslinking is a great tool to study structural changes and dynamics of ribonucleoparticles. It complements current structural biology methods like X-ray crystallography and NMR and will be highly useful in many research areas, from basic molecular biology to drug design. Finally, we believe it is possible and feasible to extend our protein:RNA approach to study protein:DNA interactions, something that will be of great interest to a wide audience of researchers.
For protein:RNA interactions, we sought to develop a means for the identification of the peptide, and the exact amino acid that is crosslinked to RNA during UV irradiation in living cells. This is based on the identification of the specific mass difference resulting from the single residual nucleotide that remains associated with the peptide following complete hydrolysis of the RNA and partial digestion of the protein. We precisely mapped the RNA-binding sites of hundreds of yeast proteins following crosslinking in actively growing cells, validated by using available high-resolution structures for yeast ribosome and individual protein-RNA complexes, together with genome-wide analysis of RNA targets of the novel RNA-binding protein enolase. This approach should be widely applicable for the reliable and accurate characterization of the protein-RNA interactome in many systems. We are applying it to ribosome biology at the moment and developing it further. Protein:protein crosslinking was applied to study the changes in composition and structure of Escherichia coli ribosomes from ribosome assembly-deficient strains. First results confirmed applicability of the proposed methods for the task, further work is in progress.
The combination of protein:RNA and protein:protein crosslinking is a great tool to study structural changes and dynamics of ribonucleoparticles. It complements current structural biology methods like X-ray crystallography and NMR and will be highly useful in many research areas, from basic molecular biology to drug design. Finally, we believe it is possible and feasible to extend our protein:RNA approach to study protein:DNA interactions, something that will be of great interest to a wide audience of researchers.