Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase

Nucleic Acids Research Oxford University Press (2019) gkz797

TD Craggs, M Sustarsic, A Plochowietz, M Mosayebi, H Kaju, A Cuthbert, J Hohlbein, L Domicevica, PC Biggin, J Doye, A Kapanidis

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA-Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA-Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1-2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

Tracking antibiotic mechanisms.

Nature reviews. Microbiology 17 (2019) 201-201

OJ Pambos, AN Kapanidis

Single Nitrogen-Vacancy Imaging in Nanodiamonds for Multimodal Sensing

BIOPHYSICAL JOURNAL 116 (2019) 174A-174A

M Sow, H Steuer, B Gilboa, L Gines, S Mandal, S Adekanye, JM Smith, OA Williams, AN Kapanidis

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics

Nucleic Acids Research Oxford University Press (OUP) (0)

NC Robb, AJW te Velthuis, E Fodor, AN Kapanidis

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes.

Microbial cell (Graz, Austria) 6 (2019) 65-101

HL Klein, KKH Ang, MR Arkin, EC Beckwitt, Y-H Chang, J Fan, Y Kwon, MJ Morten, S Mukherjee, OJ Pambos, H El Sayyed, ES Thrall, JP Vieira-da-Rocha, Q Wang, S Wang, H-Y Yeh, JS Biteen, P Chi, W-D Heyer, AN Kapanidis, JJ Loparo, TR Strick, P Sung, B Van Houten, H Niu, E Rothenberg

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Recent Advances in Understanding σ70-Dependent Transcription Initiation Mechanisms.

Journal of molecular biology (2019)

A Mazumder, AN Kapanidis

Prokaryotic transcription is one of the most studied biological systems, with relevance to many fields including the development and use of antibiotics, the construction of synthetic gene networks, and the development of many cutting-edge methodologies. Here, we discuss recent structural, biochemical, and single-molecule biophysical studies targeting the mechanisms of transcription initiation in bacteria, including the formation of the open complex, the reaction of initial transcription, and the promoter escape step that leads to elongation. We specifically focus on the mechanisms employed by the RNA polymerase holoenzyme with the housekeeping sigma factor σ70. The recent progress provides answers to long-held questions, identifies intriguing new behaviours, and opens up fresh questions for the field of transcription.

Confinement-free wide-field ratiometric tracking of single fluorescent molecules

Biophysical Journal Elsevier BV (2019)

B Gilboa, B Jing, TJ Cui, M Sow, A Plochowietz, A Mazumder, AN Kapanidis

Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction

Scientific Reports Nature Research 9 (2019) 16219

N Robb, J Taylor, A Kent, A Kapanidis, O Pambos, B Gilboa

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

Nature Communications Nature Publishing Group 9 (2018) Article number 1478-

D Dulin, D Bauer, A Malinen, J Bakermans, M Kaller, Z Morichaud, I Petushkov, M Depken, K Brodolin, A Kulbachinskiy, A Kapanidis

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Molecular cell (2018)

W Lin, K Das, D Degen, A Mazumder, D Duchi, D Wang, YW Ebright, RY Ebright, E Sineva, M Gigliotti, A Srivastava, S Mandal, Y Jiang, Y Liu, R Yin, Z Zhang, ET Eng, D Thomas, S Donadio, H Zhang, C Zhang, AN Kapanidis, RH Ebright

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

Tracking tRNA packages.

Nature chemical biology 14 (2018) 528-529

AN Kapanidis, M Stracy

Wide-Field Monitoring of Single Fluorescent Molecules and Nanoparticles without Immobilization

BIOPHYSICAL JOURNAL 114 (2018) 169A-169A

B Gilboa, B Jing, M Sow, TJ Cui, A Plochowietz, AN Kapanidis

The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp.

Nucleic acids research 46 (2018) 7284-7295

D Duchi, A Mazumder, AM Malinen, RH Ebright, AN Kapanidis

RNA polymerase (RNAP) contains a mobile structural module, the 'clamp,' that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including promoter melting, transcription elongation complex stability, transcription pausing, and transcription termination. Using single-molecule FRET on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme populates three distinct conformational states and interconvert between these states on the 0.1-1 s time-scale. Similar studies confirm that the RNAP clamp is closed in open complex (RPO) and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, the clamp does not exhibit dynamic behaviour. We also show that, the stringent-response alarmone ppGpp, which reprograms transcription during amino acid starvation stress, selectively stabilizes the partly-closed-clamp state and prevents clamp opening; these results raise the possibility that ppGpp controls promoter opening by modulating clamp dynamics.

Short-Read Single-Molecule DNA Sequencing for Highly Parallel Analysis of Protein-DNA Interactions


R Andrews, H Steuer, A Shivalingam, AH El-Sagheer, T Brown, AN Kapanidis

Single-molecule analysis of the influenza virus replication initiation mechanism

Biophysical Journal Biophysical Society 114 (2018) 246A-246A

N Robb, AJW te Velthuis, E Fodor, A Kapanidis

Coming together during viral assembly.

Nature reviews. Microbiology 16 (2018) 721-721

C Hepp, NC Robb

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nature methods 15 (2018) 669-676

B Hellenkamp, S Schmid, O Doroshenko, O Opanasyuk, R Kühnemuth, S Rezaei Adariani, B Ambrose, M Aznauryan, A Barth, V Birkedal, ME Bowen, H Chen, T Cordes, T Eilert, C Fijen, C Gebhardt, M Götz, G Gouridis, E Gratton, T Ha, P Hao, CA Hanke, A Hartmann, J Hendrix, LL Hildebrandt, V Hirschfeld, J Hohlbein, B Hua, CG Hübner, E Kallis, AN Kapanidis, J-Y Kim, G Krainer, DC Lamb, NK Lee, EA Lemke, B Levesque, M Levitus, JJ McCann, N Naredi-Rainer, D Nettels, T Ngo, R Qiu, NC Robb, C Röcker, H Sanabria, M Schlierf, T Schröder, B Schuler, H Seidel, L Streit, J Thurn, P Tinnefeld, S Tyagi, N Vandenberk, AM Vera, KR Weninger, B Wünsch, IS Yanez-Orozco, J Michaelis, CAM Seidel, TD Craggs, T Hugel

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes

Nucleic Acids Research 46 (2018) 677-688

D Duchi, K Gryte, NC Robb, Z Morichaud, C Sheppard, K Brodolin, S Wigneshweraraj, AN Kapanidis

© The Author(s) 2017. Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex sub-sequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia colitranscription open complexes. Our results revealed the existence of two stable states within RNAP-DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nature methods 15 (2018) 984-984

B Hellenkamp, S Schmid, O Doroshenko, O Opanasyuk, R Kühnemuth, SR Adariani, B Ambrose, M Aznauryan, A Barth, V Birkedal, ME Bowen, H Chen, T Cordes, T Eilert, C Fijen, C Gebhardt, M Götz, G Gouridis, E Gratton, T Ha, P Hao, CA Hanke, A Hartmann, J Hendrix, LL Hildebrandt, V Hirschfeld, J Hohlbein, B Hua, CG Hübner, E Kallis, AN Kapanidis, J-Y Kim, G Krainer, DC Lamb, NK Lee, EA Lemke, B Levesque, M Levitus, JJ McCann, N Naredi-Rainer, D Nettels, T Ngo, R Qiu, NC Robb, C Röcker, H Sanabria, M Schlierf, T Schröder, B Schuler, H Seidel, L Streit, J Thurn, P Tinnefeld, S Tyagi, N Vandenberk, AM Vera, KR Weninger, B Wünsch, IS Yanez-Orozco, J Michaelis, CAM Seidel, TD Craggs, T Hugel

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Understanding Protein Mobility in Bacteria by Tracking Single Molecules.

Journal of molecular biology 430 (2018) 4443-4455

AN Kapanidis, S Uphoff, M Stracy

Protein diffusion is crucial for understanding the formation of protein complexes in vivo and has been the subject of many fluorescence microscopy studies in cells; however, such microscopy efforts are often limited by low sensitivity and resolution. During the past decade, these limitations have been addressed by new super-resolution imaging methods, most of which rely on single-particle tracking and single-molecule detection; these methods are revolutionizing our understanding of molecular diffusion inside bacterial cells by directly visualizing the motion of proteins and the effects of the local and global environment on diffusion. Here we review key methods that made such experiments possible, with particular emphasis on versions of single-molecule tracking based on photo-activated fluorescent proteins. We also discuss studies that provide estimates of the time a diffusing protein takes to locate a target site, as well as studies that examined the stoichiometries of diffusing species, the effect of stable and weak interactions on diffusion, and the constraints of large macromolecular structures on the ability of proteins and their complexes to access the entire cytoplasm.