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 platform1–3. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses5,6 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.

DNA polymerase β (Pol β) is a 39 kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers and these mutations further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s–1. Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped, DNA substrate bound form. Upon binding DNA, these lyase domain motions are significantly quenched whereas evidence for conformational motions in the polymerase domain become apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation, lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity.

Riboswitches is RNA structural elements generally located in the 5ˈuntranslated region (3ˈUTR) of mRNA. In the genetic regulation, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis for this mechanism requires the ability to study structural changes over time. We apply 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 prior to diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction involving two apo, one ligand-bound intermediate, and the final bound states. These structures support a reaction mechanism model with at least four states and illustrate the structural basis for signal transmission. The two apo conformers differ significantly in the three-way junction and the P1 switch helix relative to 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 a >10-minute delay, the RNA molecules were fully converted to the bound state, in which the substantial conformational changes resulted in conversion of the space group. Such drastic changes in crystallo highlight the important opportunities that micro/nanocrystals may offer in these and similar time-resolved diffraction studies. These results all together demonstrate the potential of "mix-and-inject" time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those involving large conformational changes.

DNA polymerase β (Pol β) repairs single-nucleotide gapped DNA (sngDNA) by enzymatic incorporation of the Watson-Crick partner nucleotide at the gapped position opposite the templating nucleotide. The process by which the matching nucleotide is incorporated into a sngDNA sequence has been relatively well-characterized, but the process of discrimination from nucleotide misincorporation remains unclear. We report here NMR spectroscopic characterization of full-length, uniformly labeled Pol β in apo, sngDNA-bound binary, and ternary complexes containing matching and mismatching nucleotide. Our data indicate that, while binding of the correct nucleotide to the binary complex induces chemical shift changes consistent with the process of enzyme closure, the ternary Pol β complex containing a mismatching nucleotide exhibits no such changes and appears to remain in an open, unstable, binary-like conformation. Our findings support an induced-fit mechanism for polymerases in which a closed ternary complex can only be achieved in the presence of matching nucleotide.