title: Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid.
abstract: Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom;
authors.forename: Mathew
authors.initials: M
authors.lastname: Stracy
authors.affiliation: Bases Moléculaires et Structurales des Systèmes Infectieux, UMR 5086, Centre National de la Recherche Scientifique, University of Lyon, 69 367 Lyon, France;
authors.forename: Christian
authors.initials: C
authors.lastname: Lesterlin
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom;
authors.forename: Federico
authors.initials: F
authors.lastname: Garza de Leon
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom; Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Stephan
authors.initials: S
authors.lastname: Uphoff
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom; Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
authors.forename: Pawel
authors.initials: P
authors.lastname: Zawadzki
authors.affiliation: Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom; a.kapanidis1@physics.ox.ac.uk.
authors.forename: Achillefs N
authors.initials: AN
authors.lastname: Kapanidis
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D050296:Microbial Viability / Q000187:drug effects; D008853:Microscopy / Q000379:methods*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D012293:Rifampin / Q000494:pharmacology; D059008:Time-Lapse Imaging; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin
keywords: RNA polymerase; protein-DNA interactions; single-molecule tracking; superresolution; transcription