Biology subjects

America, P.

Publications and source records attributed to America, P..

6 recordsLinked to original sources

bioRxiv2026

RfaH licenses RNA polymerase for long-range transcription

Processivity is essential for gene expression: Escherichia coli RNA polymerase (RNAP) must transcribe 10+ kbp operons without failure, yet backtrack-induced long-lived pauses threaten premature termination. Gre factors stimulate transcript cleavage to rescue backtracked RNAP, whereas NusA and NusG, which bridge the expressome, respectively stimulate and suppress pausing. How they cooperate to secure full-length transcription is unclear. Using high-throughput magnetic tweezers, we reconstitute ops-induced pausing, showing that sequence context sets pause occupancy. RfaH, a NusG paralog recruited at ops and essential for long virulence operons, loads onto ops-paused RNAP by two pathways: one permitting immediate escape, the other requiring GreA rescue. We find that both NusG and RfaH nullify NusA's pause-stimulating effect, RfaH sustaining runs four times longer than NusG and replicating its role in vivo, where RfaH depletion leads to conjugation failing. This work provides the foundation for dissecting virulence operon expression and its therapeutic targeting.

biophysics↗

bioRxiv2026

Processing, analysing and modelling kinetic data in the era of high-throughput single-molecule biophysics

Biomolecular reactions are often composed of multiple stochastic, reversible and branched transition paths over intermediates, leading to rich dynamics. Single-molecule biophysics has revolutionized our view of biology by revealing the heterogeneity in realized paths and pointing to the importance of rare events. The recent development of high-throughput single-molecule biophysics techniques now allow to quantitatively study this heterogeneity and characterize even the rarest kinetic events. Processing, analyzing and modelling high-throughput single-molecule data has been the focus of several reports, but are often difficult to implement for non-experts. Here, we provide a guide to extract the most from transitions in single-molecule biophysics data using a first-passage time framework and maximum likelihood estimation. We specifically focused on parameter sweeps in systems with one or two characteristic timescales, and show how they can be analyzed in terms of a minimal kinetic model and its dependence on enzyme/substrate concentration, force and temperature. We introduce a general framework to perform data-driven modelling on single- and two-state models and illustrate it with concrete examples. We also provide programs with graphical user interfaces to perform such analysis on raw data, in the hope that it will empower experimental single-molecule biophysicists to extract the most out of their data.

biophysics↗

bioRxiv2026

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

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.

biophysics↗

bioRxiv2025

Template switching by coronavirus polymerase requires helicase activity and is stimulated by remdesivir and molnupiravir

Polymerase template switching is an essential mechanism in coronaviruses (CoVs) that enables both sub-genomic (sg) RNA synthesis and increases genomic diversity via RNA recombination. Despite its importance, the molecular mechanism of CoV polymerase template switching remains unclear. Using magnetic tweezers, we show that the CoV non-structural protein (nsp) 13-helicase drives polymerase template switching, followed by copy-back RNA synthesis. This activity requires nsp13-helicase ATPase activity and a duplex RNA downstream of the CoV polymerase. This novel function of nsp13-helicase is targeted by the nucleotide analogs remdesivir and molnupiravir, whose incorporation in the nascent strand increases CoV polymerase template switching probability, leading to defective RNA production. We propose a novel mechanism of action where incorporation of these analogs dramatically reduces full length genome copy number by stimulating polymerase template switching. Our study further demonstrates nsp13-helicases central role in CoV replication and how this enzyme function can be indirectly targeted by analogs.

microbiology↗

bioRxiv2025

The coronavirus helicase synergizes with the viral RNA polymerase to enable rapid RNA synthesis through duplex RNA

Positive-sense (+) RNA viruses often encode helicases presumed to support replication. Their precise role remains unresolved though, especially in coronaviruses (CoV) where the helicase translocates in the opposite direction to the polymerase. Using high-throughput single-molecule magnetic tweezers, we show that the coronavirus helicase enhances RNA synthesis through duplex RNA by tenfold, forming a directional complex with the viral polymerase. Despite opposing polarity, the helicase coordinates elongation by engaging the non-template strand. A detailed kinetic model derived from large datasets reveals distinct dynamic states, including fast bursting and slow, backtracking-prone modes, which are governed by helicase engagement. These results uncover an active coupling mechanism that modulates replication dynamics and provide a mechanistic basis for continuous versus discontinuous RNA synthesis in coronaviruses. Our findings establish the viral helicase as a central regulator of RNA replication rather than a passive accessory enzyme.

biophysics↗

bioRxiv2024

Single-molecule magnetic tweezers reveals that TAV2b-derived peptides underwind and stabilize double-stranded RNA

Double-stranded RNA (dsRNA) has evolved into a key tool in understanding and regulating biological processes, with promising implications in therapeutics. However, its efficacy is often limited due to instability in biological settings. Recently, the development of peptidic dsRNA binders derived from naturally occurring RNA-binding proteins has emerged as a favorable starting point to address this limitation. Nevertheless, it remains unclear how these high affinity dsRNA binders alter the structure and flexibility of dsRNA. To this end, we employed single-molecule magnetic tweezers experiments to investigate the effects of TAV2b-derived peptidic dsRNA binders on the mechanical properties of dsRNA. Torque spectroscopy assays demonstrated that these peptides underwind dsRNA, while also stabilizing the duplex. Additionally, force spectroscopy experiments demonstrate that a wild type TAV2b peptide derivative extends the contour length and lowers the bending rigidity of dsRNA, while a homodimeric version triggers the formation of higher order complexes at forces below 1 pN. Our study presents a quantitative approach to investigate how these peptides alter the structure of dsRNA, and whether peptide structural design alters the affinity to dsRNA and its stability. This approach could inform the design of more potent and effective dsRNA binders in the efforts to advance RNA therapeutics.

biophysics↗