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Proc Natl Acad Sci U S A. 2015 Aug 11;112(32):E4390-9. doi: 10.1073/pnas.1507592112. Epub 2015 Jul 29.

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid.

Author information

1
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom;
2
Bases Moléculaires et Structurales des Systèmes Infectieux, UMR 5086, Centre National de la Recherche Scientifique, University of Lyon, 69 367 Lyon, France;
3
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.
4
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom; a.kapanidis1@physics.ox.ac.uk.

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.

KEYWORDS:

RNA polymerase; protein-DNA interactions; single-molecule tracking; superresolution; transcription

PMID:
26224838
PMCID:
PMC4538611
DOI:
10.1073/pnas.1507592112
[Indexed for MEDLINE]
Free PMC Article

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