RSDyn · Ribosome dynamics analysed by novel cross-linking/mass spectrometry
7РП — „Хора“ (Действия „Мария Кюри“)
- Период
- 2012-12-01 → 2014-11-30
- Финансиране от ЕС
- 209 033 €
- Участници
- 1
- Схема
- MC-IEF
Линиите свързват координатора с партньорите.
Накратко на български
Динамиката на рибозомите се анализира чрез проследяване на връзките между протеини и РНК, например при дрожди и бактерии E. coli. Това помага за по-точното разбиране на структурата на рибонуклеопротеините, което е полезно за молекулярната биология и разработването на лекарства.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
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.
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
Ribosomes, large and dynamic particles made of RNA and proteins, are amongst the most complex macromolecular machines known to man. High resolution tools have provided exciting views of the ribosome structure. Revealing dynamics of ribosomal complexes in solution, however, has so far been challenging due to the restricted stability and homogeneity of ribosomes. A set of new tools developed by the Rappsilber lab, partly by me over the last few months, changes dramatically the feasibility of investigating ribosome dynamics. I aim to use these new mass spectrometry-based technologies to determine accurate sites and changes in yield of cross-linking between proteins and between RNA and proteins. Thereby, using protein-protein and RNA-protein cross-linking/mass spectrometry approaches, I aim to answer:1.Is the final and rate-limiting step in the biogenesis of 50S ribosomal subunits a conformational rearrangement? This will prepare the subsequent analysis of the other steps during the biogenesis of ribosomes in E. coli and in future also in the eukaryote S. cerevisiae.2.How does the structure and extra-ribosomal protein content of translating ribosomes change in response to different stress conditions? This will complement electron microscopy data and lead into the analysis of how translation links to virulence in pathogenic bacteria.My project will showcase the potential of cross-linking/mass spectrometry for the study of ribonucleoprotein complexes and, indeed, for protein-nucleotide interactions in general, i.e. encompass DNA-protein interactions. Furthermore, results of this study may facilitate the development of next-generation antibacterial drugs, targeting assembly factors and conformational rearrangements during ribosome biogenesis. For my academic career, acquiring knowledge in cutting edge proteomics approaches will be a stepping stone and further strengthen my role as gatekeeper of proteomics knowledge and a catalyst of biological sciences in Estonia.
Оригинален текст от CORDIS (на английски).
Участници
- THE UNIVERSITY OF EDINBURGH · EdinburghКоординаторОбединеното кралство
Връзки
Данни: CORDIS, © Европейски съюз