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MIRNA Informe resumido

Project ID: 259092
Financiado con arreglo a: FP7-IDEAS-ERC
País: Switzerland

Final Report Summary - MIRNA (Metal Ions and Metal Ion Complexes Guiding Folding and Function of Single RNA Molecules)

Metal ions are instrumental to folding, structure and function of nucleic acids, and in particular also of larger RNAs like ribozymes and riboswitches. This project aims to elucidate and understand the specific role of metal ions and metal ion complexes on the single molecule level. We have concentrated mainly on two classes of functional RNAs, riboswitches and self-splicing group II intron ribozymes.

Riboswitches are important regulatory elements (mostly) in bacteria, acting either on the translational or the transcriptional level. We could show that different regions in the adenosyl cobalamin riboswitch btuB from E. coli exhibit different dissociation constants towards Mg2+ indicating a stepwise folding. Above five mM Mg2+, the binding competent confirmation is reached, being driven further towards the switched form by higher Mg2+ concentration. At the same time, Tb(III) cleavage assays revealed crucial Mn+ binding sites near tertiary contacts and the ligand binding site. To further elucidate the folding, binding, and switching mechanism, a number of light stable and fluorophore carrying B12 derivatives have been synthesized. Single molecule studies are ongoing to investigate in detail the folding pathway and structural rearrangement of the RNA upon metabolite binding. In order to carry out such detailed investigations, a Mathlab-based software MASH has been developed in our lab, allowing for a fast, efficient, and state-of-the-art processing and evaluation of smFRET data. This proved crucial to understand the role of metal ions on the atomic level with regard to their coordination chemical properties. The new software also includes a statistical bootstrap-based analysis tool (BOBA-FRET), which is unprecedented in the smFRET field, but allows us to add error limits and confidence values to the identified FRET values. Both software tools are available for free from our lab.

Studies on a large RNA, the group II intron ribozyme, being phylogenetic precursor to the largest part of the human genome, have revealed numerous important results: (i) RNA-RNA interactions are directly dependent on binding to divalent metal ions, the individual affinities for phosphate groups and ring nitrogens determining on- and off rates, and the affinity according to the Irving-Williams-Series. (ii) Heterogeneity of RNA-RNA interactions is directly dependent on the loading factor of metal ion binding sites. (iii) The addition of crowing agents like PEG, strongly reduces the requirement for divalent metal ions and leads to an active and more compact ribozyme both in bulk and on the single molecule level. (iv) By introducing specific point mutations to inhibit tertiary contact, we can a distinct folding order of the individual domains to the various FRET states. (v) A novel labelling technique for large RNAs has been developed in collaboration with Dr. Eva Freisinger, allowing to place a fluorophore site-specifically within large RNAs. Taken together, highly important results and crucial progress has been achieved at the interface between Nucleic Acids Chemistry, Biophysics, and Bioinorganic Chemistry.

Contacto

Roland Karl Oliver Sigel, (Professor)
Tel.: +41446354652
Fax: +41446356802
Correo electrónico
Número de registro: 183377 / Última actualización el: 2016-05-26
Fuente de información: SESAM