Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography Academic Article uri icon

abstract

  • Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

authors

  • Stagno, JR
  • Liu, Y
  • Bhandari, YR
  • Conrad, CE
  • Panja, S
  • Swain, M
  • Fan, L
  • Nelson, G
  • Li, C
  • Wendel, DR
  • White, TA
  • Coe, JD
  • Wiedorn, MO
  • Knoska, J
  • Oberthuer, D
  • Tuckey, RA
  • Yu, P
  • Dyba, M
  • Tarasov, SG
  • Weierstall, U
  • Grant, TD
  • Schwieters, CD
  • Zhang, J
  • Ferré-D’Amaré, AR
  • Fromme, P
  • Draper, DE
  • Liang, M
  • Hunter, MS
  • Boutet, S
  • Tan, K
  • Zuo, X
  • Ji, X
  • Barty, A
  • Zatsepin, NA
  • Chapman, HN
  • Spence, JCH
  • Woodson, SA
  • Wang, Y-X

publication date

  • January 2017

published in