Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins.

Molecular cell 81:7 (2021) 1499-1514.e6

Authors:

Mathew Stracy, Jakob Schweizer, David J Sherratt, Achillefs N Kapanidis, Stephan Uphoff, Christian Lesterlin

Abstract:

Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Toward identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid is not a physical barrier for protein diffusion but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58%-99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome crowding likely has important implications for the function of all DNA-binding proteins.

RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding.

Nucleic acids research 49:5 (2021) 2790-2802

Authors:

Abhishek Mazumder, Anna Wang, Heesoo Uhm, Richard H Ebright, Achillefs N Kapanidis

Abstract:

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

Amplification-Free Detection of Viruses in Minutes using Single-Particle Imaging and Machine Learning

BIOPHYSICAL JOURNAL 120:3 (2021) 195A-195A

Authors:

Nicolas Shiaelis, Leon Peto, Andrew McMahon, Chritof Hepp, Erica Bickerton, Cyril Favard, Delphine Muriaux, Monique Andersson, Alison Vaughan, Philippa Matthews, Nicole Stoesser, Derrick Crook, Achillefs N Kapanidis, Nicole C Robb

Closing and opening of the RNA polymerase trigger loop

Proceedings of the National Academy of Sciences National Academy of Sciences 117:27 (2020) 15642-15649

Authors:

Abhishek Mazumder, Miaoxin Lin, Achillefs N Kapanidis, Richard H Ebright

Abstract:

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

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

Nucleic Acids Research Oxford University Press (2019) gkz797

Authors:

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

Abstract:

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.