Controllable molecular motors engineered from myosin and RNA

Abstract

Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems1 or in living cells2. Previously, synthetic nucleic acid motors3–5 and modified natural protein motors6–10 have been developed in separate complementary strategies for achieving tunable and controllable motor function. Integrating protein and nucleic acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number, and composition of tethered protein motors11–15. Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport, and can be dynamically controlled using programmed transitions in lever arm structure7,9. We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy, and structural probing16. Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement17 reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10–20 nm s−1. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

The energy landscape of −1 ribosomal frameshifting

“…A systematic study of different frameshifting elements may reveal their effect on the energy landscape of frameshifting delineated here as well as distinctive frameshifting pathways used at other stages of translation. A deep understanding of frameshifting mechanisms may permit its use in artificial regulation of gene expression at the translation level through external cues such as temperature or oligonucleotide signals ( 43 ).…”

Cryo-EM structures reveal specialization at the myosin VI-actin interface and a mechanism of force sensitivity

“…We utilized an engineered myosin VI construct comprising the MD with 1 IQ fused to an RNA-binding L7Ae kink-turn domain ( Figure 1—figure supplement 1A,C ). The L7Ae kink-turn domain is oriented such that RNA-binding extends the lever arm and can tune motor activity ( Omabegho et al, 2017 ). Combining datasets with and without RNA bound improved the resolution of our reconstruction considerably, suggesting that RNA binding does not alter motor conformation ( Figure 1—figure supplement 1B ).…”

“…Myosin VI was engineered and purified as previously described ( Omabegho et al, 2017 ). Briefly, a DNA construct for protein expression was assembled from fragments encoding porcine myosin VI (residues 1–817) and Archaeoglobus fulgidus L7Ae (residues 9–118), cloned into a pBiex-1 (Novagen-Millipore, Burlington, MA) expression vector modified to include codons for a C-terminal eYFP, and FLAG tag (DYKDDDDK) with intervening GSG repeats (see Figure 1—figure supplement 1 ).…”

The C-terminal actin binding domain of talin forms an asymmetric catch bond with F-actin

“…Actin filament polarity was explicitly determined as described in Huang et al, 2017 for a subset of the data in which flow cells were prepared with a myosin VI construct consisting of porcine myosin VI (residues 1-817) and Archaeoglobus fulgidus L7Ae (residues 9-118) with a C-terminal eYFP (Omabegho et al, 2018) on one half of the flow cell (Huang et al, 2017). In this case, actin filaments were held above multiple myosin VI platforms and the direction of myosin force generation was observed.…”