Visualizing transient low-populated structures of RNA

Journal name:
Nature
Volume:
491,
Pages:
724–728
Date published:
DOI:
doi:10.1038/nature11498
Received
Accepted
Published online

Abstract

The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized so far involve long-lived species that can be captured by structure characterization techniques. Here we report the nuclear magnetic resonance visualization of RNA transitions towards ‘invisible’ excited states (ESs), which exist in too little abundance (2–13%) and for too short a duration (45–250μs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix–junction–helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signalling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes.

At a glance

Figures

  1. Excited-state structure of the HIV-1 TAR apical loop.
    Figure 1: Excited-state structure of the HIV-1 TAR apical loop.

    a, GS and ES structures of TAR. ES chemical shifts indicating increased stacking and/or anti glycosidic angles and C3′-endo sugar pucker are blue; decreased stacking and/or syn glycosidic angles and non-C3′-endo sugar pucker are red. Sites with little to no fast exchange are grey. b, Example relaxation dispersion profile showing dependence of R2 + Rex on spin-lock power (ωeff/2π) and offset (Ω/2π), where Ω is the difference between the observed resonance frequency and the spin-lock carrier frequency. Shown is a global fit (solid line) to a two-state Laguerre equation. Error bars indicate one s.d. c, Mutant mimics of GS and ES. d, Comparison of carbon chemical shifts (CS) for the ES, GS and mutant mimics. Carbons at the site of mutation are indicated using an asterisk. e, Proposed functional role for TAR ES.

  2. Excited-state structure of the ribosomal A-site internal loop.
    Figure 2: Excited-state structure of the ribosomal A-site internal loop.

    a, GS and ES structures of the A-site. Chemical shift fingerprints are colour-coded as in Fig. 1a. b, Example relaxation dispersion profile (as in Fig. 1b). c, Mutant mimics of GS and ES. d, Comparison of carbon chemical shifts for the ES, GS and mutant mimics. e, Proposed functional role for A-site ES.

  3. Two mutually exclusive excited-state structures in HIV-1 stem-loop 1.
    Figure 3: Two mutually exclusive excited-state structures in HIV-1 stem-loop 1.

    a, GS and ES structures of SL1m. Chemical shift fingerprints are colour-coded as in Fig. 1a. b, Example relaxation dispersion profile (as in Fig. 1b). c, Mutant mimics of GS and ES. d, Comparison of carbon chemical shifts for the ESs, GS and mutant mimics. e, Proposed mechanism for spontaneous kissing-duplex isomerization. f, Native gel showing the reduction in isomerization rate caused by inhibiting exchanging conformations (see Supplementary Fig. 8).

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Author information

  1. These authors contributed equally to this work.

    • Elizabeth A. Dethoff,
    • Katja Petzold &
    • Jeetender Chugh

Affiliations

  1. Department of Chemistry & Biophysics, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, USA

    • Elizabeth A. Dethoff,
    • Katja Petzold,
    • Jeetender Chugh,
    • Anette Casiano-Negroni &
    • Hashim M. Al-Hashimi
  2. Present address: NYMIRUM, 3510 West Liberty Road, Ann Arbor, Michigan 48103, USA.

    • Anette Casiano-Negroni

Contributions

H.M.A., E.A.D., K.P. and J.C. conceived the approaches to structurally characterize RNA ES and wrote the paper. E.A.D. and K.P. performed all experiments and data analyses for HIV TAR and SL1m, respectively. J.C. with assistance from A.C.-N. performed all experiments and data analyses for the A-site.

Competing financial interests

H.M.A. is an advisor to and holds an ownership interest in Nymirum Inc., which is an RNA-based drug discovery company. The research reported in this article was performed by the University of Michigan faculty and students and was funded by an NIH contract to H.M.A.

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    This file contains Supplementary Figures 1-8, Supplementary Tables 1-2, a Supplementary Discussion and additional references.

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