Accurate single-bead force calibration in high-throughput magnetic tweezers reveals the mechanism of directional transcription termination by MTERF1

ABSTRACT High-throughput force spectroscopy assays, such as with magnetic tweezers, enable reconstruction of biomolecular reaction energy landscapes and provide access to rare events with deep statistics. Precise force calibration is essential for accurately describing complex reactions, which can be hindered by sample heterogeneity, such as bead-to-bead difference in magnetic content. Here, we describe an in-situ force calibration methodology for high-throughput magnetic tweezers that enables the calibration for each individual bead with an accuracy of up to 3%, limited only by the statistical resolution. We apply this approach to characterize the directional transcription termination molecular mechanism by the polar roadblock mitochondrial transcription termination factor 1 (MTERF1). Establishing a SpyTag-SpyCatcher surface-attachment strategy, we performed force-jump experiments on the same tethers for up to 11 hours. We showed that directional DNA unwinding is sufficient to explain the polar roadblock activity of MTERF1. Accurate force spectroscopy further reveals that the unlocking transition is rate-limited by a single kinetic barrier, with a transition-state distance consistent with structural interpretations. Together, these results provide a mechanistic and broadly applicable model for the asymmetric stability of MTERF1 and other nucleic acid polar roadblocks and establish a robust force spectroscopy framework for high-throughput magnetic tweezers experiments. Competing Interest Statement The authors have declared no competing interest.