Blanc Inter II SIMI 7 - Blanc International II - SIMI 7 - Chimie moléculaire, organique, de coordination, catalyse et chimie biologique

Investigating RNA folding and chaperone activity by multidimensional NMR spectroscopy – RNAfolding

Folding and dynamics of RNA, a molecular key player in the cell

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Understanding riboswitch function and RNA-chaperone protein interactions

The aim of this project is to provide a better understanding of the folding free-energy landscape of RNA, and to show how the potential of adopting different folds of similar free energy is exploited by the RNA to perform its biological function. Our study focuses on two particular questions: (1) what are the molecular bases for the conformational transitions that riboswitch RNAs undergo as a response to changing environmental conditions in order to perform a particular function, e. g. induce or abolish gene expression? (2) What are the molecular mechanisms governing the activity of RNA chaperone proteins that assist in the folding process of RNA?

To reach our objectives, we use and further develop high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy methods to study the processes of RNA folding, ligand binding, and ligand-induced conformational transitions. The experimental NMR work is aided by the development and use of site-specific 13C isotope-labeling that provides the high spectral resolution required for NMR studies of larger RNA molecules, and ensures the availability of a large number of sensitive probes that report on the local structure and dynamics in the RNA during a refolding event, or in the presence/ absence of a metabolite ligand or chaperone protein.
The project combines state-of-the-art techniques in NMR spectroscopy, synthetic organic chemistry, and molecular biology in a multi-disciplinary international collaboration of 3 research groups.

Not yet

To early

The project has not yet reached the state of producing final results!

The historical understanding that biomolecular function is encoded in the static three-dimensional structures of proteins and nucleic acids interacting with each other is seriously being questioned. It is now well accepted that proteins often do not fold into a single conformation but exist as an ensemble of structurally distinct sub-states. Proteins seem to have evolved to establish very diverse conformational ensembles with efficient sampling of the relevant conformational space. Ligand binding leads to a redistribution of the populations, and the ultimate selection of the ‘bound’ conformation that is complementary to the interaction partner. Although there is ample experimental evidence for this “new structural biology” in the field of proteins significantly less studies have been performed in the field of nucleic acids. While X-ray crystallography has provided tremendous insight into the structural details of fundamental biological macromolecules in the past it is already foreseeable that NMR spectroscopy will play a major role in contributing experimental data that provide a comprehensive view of the structural dynamics of these molecules.
The aim of this project is to provide a better understanding of the folding free-energy landscape of RNA, and to show how the potential of adopting different folds of similar free energy is exploited by the RNA to perform its biological function. Our study will focus on two particular questions: (1) what are the molecular bases for the conformational transitions that riboswitch RNAs undergo as a response to changing environmental conditions in order to perform a particular function, e. g. induce or abolish gene expression? (2) What are the molecular mechanisms governing the activity of RNA chaperone proteins that assist in the folding process of RNA? For this purpose, we will use and further develop high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy methods to study the processes of RNA folding, ligand binding, and ligand-induced conformational transitions in real time. This will yield atomic-resolution information on the kinetics of the structural transitions, and the transient population of eventual intermediate states. In addition, we will investigate the presence of low populated excited state conformations under equilibrium conditions using spin relaxation and hydrogen exchange NMR experiments. The experimental NMR work will be aided by the development and use of site-specific 13C and 19F isotope-labeling that provide the high spectral resolution required for NMR studies of larger RNA molecules, and will ensure the availability of a large number of sensitive probes that report on the local structure and dynamics in the RNA during a refolding event, or in the presence/ absence of a metabolite ligand or chaperone protein. We have selected a number of target RNA molecules, and an RNA chaperone protein that will be produced, isotope-labeled and investigated by NMR spectroscopy during this project.
We expect that this project at the interface of NMR spectroscopy, synthetic organic chemistry, and molecular biology, and based on an international collaboration of 3 research groups that are internationally recognized experts in their respective research field, will provide new insights into the mechanistic details of RNA riboswitch function, RNA- chaperone protein interactions, and the relevance of conformational dynamics and transition states for RNA folding and function.

Project coordinator

Monsieur Bernhard BRUTSCHER (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES) – Bernhard.brutscher@ibs.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IBS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES

Help of the ANR 253,880 euros
Beginning and duration of the scientific project: January 2012 - 36 Months

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