Biology subjects

Bera, S. C.

Publications and source records attributed to Bera, S. C..

4 recordsLinked to original sources

bioRxiv2021

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor{sigma} to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RPO is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with{sigma} and the RNAP {beta} subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

biophysics↗

bioRxiv2021

Two step aggregation kinetics of Amyloid-β proteins from fractal analysis

Self-aggregation in proteins has long been studied and modeled due to its ubiquity and importance in many biological contexts. Several models propose a two step aggregation mechanism, consisting of linear growth of fibrils and branch formation. Single molecule imaging techniques such as total internal reflection fluorescence (TIRF) microscopy can provide direct evidence of such mechanisms, however, analyzing such large datasets is challenging. In this paper, we analyze for the first time, images of growing amyloid fibrils obtained from TIRF microscopy using the techniques of fractal geometry, which provides a natural framework to disentangle the two types of growth mechanisms at play. We find that after an initial linear growth phase, identified by a plateau in the average fractal dimension with time, the occurrence of branching events leads to a further increase in the fractal dimension with a final saturation value {approx} 2. We also simulate the aggregation process using the identified linear growth and secondary nucleation mechanisms, using an event driven algorithm. We theoretically model this system using a set of coupled nonlinear differential equations describing a mean field model for branching and linear growth, which we use to characterize the growth process observed in simulations as well as experiments. Finally, we provide estimates for the parameter regimes that govern the two step aggregation process observed in experiments.

biophysics↗

bioRxiv2021

The nucleotide addition cycle of the SARS-CoV-2 polymerase

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.

biophysics↗

bioRxiv2020

Signatures and mechanisms of efficacious therapeutic ribonucleotides against SARS-CoV-2 revealed by analysis of its replicase using magnetic tweezers

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. TeaserWe revise Remdesivirs mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP

biophysics↗