Single-Molecule Tracking Reveals the Functional Allocation, in vivo Interactions and Spatial Organization of Universal Transcription Factor NusG

(2022)

Authors:

Hafez El Sayyed, Oliver J Pambos, Mathew Stracy, Max Gottesman, Achillefs N Kapanidis

Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway

Journal of Molecular Biology Elsevier 434:2 (2022) 167383

Authors:

Anssi M Malinen, Jacob Bakermans, Emil Aalto-Setälä, Martin Blessing, David LV Bauer, Olena Parilova, Georgiy A Belogurov, David Dulin, Achillefs N Kapanidis

The rate-limiting step of DNA synthesis by DNA polymerase occurs in the fingers-closed conformation

Journal of Molecular Biology Elsevier 434:2 (2021) 167410

Authors:

Geraint W Evans, Timothy Craggs, Achillefs N Kapanidis

Abstract:

DNA polymerases maintain genomic integrity by copying DNA with high fidelity, part of which relies on the polymerase fingers opening-closing transition, a series of conformational changes during the DNA synthesis reaction cycle. Fingers opening and closing has been challenging to study, mainly due to the need to synchronise molecular ensembles. We previously studied fingers opening-closing on single polymerase-DNA complexes using single-molecule FRET; however, our work was limited to pre-chemistry reaction steps. Here, we advance our analysis to extensible substrates, and observe DNA polymerase (Pol) conformational changes across the entire DNA polymerisation reaction in real-time, gaining direct access to an elusive post-chemistry step rate-limiting for DNA synthesis. Our results showed that Pol adopts the fingers-closed conformation during polymerisation, and that the post-chemistry rate-limiting step occurs in the fingers-closed conformation. We found that fingers-opening in the Pol-DNA binary complex in the absence of polymerisation is slow (∼5.3 s−1), and comparable to the rate of fingers-opening after polymerisation (3.4 s−1); this indicates that the fingers-opening step itself could be largely responsible for the slow post-chemistry step, with the residual rate potentially accounted for by pyrophosphase release. We also observed that DNA chain-termination of the 3′ end of the primer increases substantially the rate of fingers-opening in the Pol-DNA binary complex (5.3 → 29 s−1), demonstrating that the 3′-OH residue is important for the kinetics of fingers conformational changes. Our observations offer mechanistic insight and tools to offer mechanistic insight for all nucleic acid polymerases.

Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

eLife eLife Sciences Publications 10 (2021) e70090

Authors:

Abhishek Mazumder, Richard H Ebright, Achillefs Kapanidis

Abstract:

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RP<sub>O</sub>). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

Viral detection and identification in 20 min by rapid single-particle fluorescence in-situ hybridization of viral RNA

Scientific Reports Springer Nature 11:1 (2021) 19579

Authors:

Christof Hepp, Nicolas Shiaelis, Nicole C Robb, Alison Vaughan, Philippa C Matthews, Nicole Stoesser, Derrick Crook, Achillefs N Kapanidis

Abstract:

The increasing risk from viral outbreaks such as the ongoing COVID-19 pandemic exacerbates the need for rapid, affordable and sensitive methods for virus detection, identification and quantification; however, existing methods for detecting virus particles in biological samples usually depend on multistep protocols that take considerable time to yield a result. Here, we introduce a rapid fluorescence in situ hybridization (FISH) protocol capable of detecting influenza virus, avian infectious bronchitis virus and SARS-CoV-2 specifically and quantitatively in approximately 20 min, in virus cultures, combined nasal and throat swabs with added virus and likely patient samples without previous purification. This fast and facile workflow can be adapted both as a lab technique and a future diagnostic tool in enveloped viruses with an accessible genome.