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Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings
Maximillian H Bailor1, Catherine Musselman1, Alexandar L Hansen1, Kush Gulati1, Dinshaw J Patel2 & Hashim M Al-Hashimi1
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1Department of Chemistry & Biophysics Research Division, The University of Michigan, Ann Arbor, Michigan 48109, USA. 2Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. Correspondence should be addressed to H.M.A. ([email protected]).
Published online 14 June 2007; doi:10.1038/nprot.2007.221
We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal WatsonCrick base pairs in helical stems are experimentally identied using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic eld. The order tensors are translated into Euler angles dening the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.
INTRODUCTIONThe functions of many regulatory RNAs involve large changes in conformation that occur in response to a range of cellular signals, including recognition of proteins and ligands, metal binding, changes in temperature and RNA synthesis itself14. Such confor
mational transitions allow one RNA molecule to carry out many biochemical transactions. For example, the RNA conformation required for the assembly of a complex ribonucleoprotein may differ from that required for executing the ribonucleoprotein function5,6. Conformational changes also provide a basis for sensing signals and transmitting regulatory responses. For example, a large class of mRNA riboswitches regulate gene expression by changing conformation in response to recognition of small meta-bolite molecules or changes in temperature7,8.
These and many other examples (see refs....