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Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

Nature Protocols volume 2, pages 1536–1546 (2007)Cite this article

Abstract

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 Watson–Crick base pairs in helical stems are experimentally identified 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 field. The order tensors are translated into Euler angles defining 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.

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Figure 1
Figure 2: Determining the relative orientation and dynamics of A-form helices using an order tensor analysis of RDCs46,99.
Figure 3: Typical RDCs measured in base and sugar moieties of RNA using the pulse sequences listed in Table 1.
Figure 4: Example illustrating application of protocol in the determination of the relative orientation and dynamics of two helices in the free state of HIV-1 TAR RNA.
Figure 5: The relative orientation (interhelix bend, θh, and twist angles, ξ) and dynamics (ϑint) of RNA helices obtained from order tensor analysis of RDCs under different contexts.

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Acknowledgements

We thank members of the Al-Hashimi laboratory for insightful comments and Dr. Alex Kurochkin for his expertise and for maintenance of the NMR instruments. H.M.A. acknowledges fruitful collaborations with the groups of Carol Fierke (The University of Michigan) and Ioan Andricioaei (The University of Michigan). We gratefully acknowledge the Michigan Economic Development Cooperation and the Michigan Technology Tri-Corridor for the support in the purchase 600 MHz spectrometer. This work was supported by funding from the NIH (RO1 AI066975-01).

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  1. Department of Chemistry & Biophysics Research Division, The University of Michigan, Ann Arbor, 48109, Michigan, USA

    Maximillian H Bailor, Catherine Musselman, Alexandar L Hansen, Kush Gulati & Hashim M Al-Hashimi

  2. Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    Dinshaw J Patel

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  1. Maximillian H Bailor
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Correspondence to Hashim M Al-Hashimi.

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Bailor, M., Musselman, C., Hansen, A. et al. Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings. Nat Protoc 2, 1536–1546 (2007). https://doi.org/10.1038/nprot.2007.221

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