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.RNA polymerase | transcription | superresolution | single-molecule tracking | protein-DNA interactions

Advances in Imaging Plant Cell Dynamics

“…Thus, live SIM studies have been conducted in bacteria (Bottomley et al, 2017), showing the subcellular distribution of RNA polymerase (Stracy et al, 2015) and the time-resolved assembly of the FtsZ ring during bacterial division (Strauss et al, 2012). 2D and 3D SIM can temporally resolve subcellular structures of small eukaryotic cells, such as the spindle pole body (Fig.…”

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

“…Consistent with the idea that at least some of these fluorescent foci represented rRNA operons, their intensities declined in starved or slowly growing cells. Superresolution imaging showed that RNAPs formed clusters that occupied specific regions of the nucleoid under the conditions examined; i.e., at fast growth rates when there are large numbers of RNAPs actively transcribing rRNA operons (Bratton et al 2011;Bakshi et al 2013;Endesfelder et al 2013;Stracy et al 2015). However, such studies directly addressed only the locations of RNAP and not the positions of the different rRNA operons.…”

Omega (ParB) binding sites together with the RNA polymerase-recognized sequence are essential for centromeric functions of the Pω region in the partition system of pSM19035

“…The importance of RNA polymerase and transcription for segregation of bacterial chromosomes has already been reported (Dworkin & Losick, 2002;Kjos & Veening, 2014;Stracy et al, 2015;Woldringh, 2002). Transcription of many genes distributed throughout the genome might provide driving force for chromosome segregation, maintain appropriate chromosomal structure with supercoils separated by expressed regions, or reorganize the nucleoid within the cell (Kjos & Veening, 2014;Le et al, 2013;Stracy et al, 2015;Woldringh, 2002). Moreover, transertion (co-transcriptional translational and protein translocation) of membrane proteins might generate DNA movement resulting from competition of DNA-RNAP-mRNA-ribosome complexes for membrane surface (Matsumoto et al, 2015;Woldringh, 2002).…”

“…Therefore, our results indicate that the recently reported (Volante et al, 2015) cooperative binding of Omega and RNA polymerase is essential not only for regulating gene transcription but also for the partition process itself. The importance of RNA polymerase and transcription for segregation of bacterial chromosomes has already been reported (Dworkin & Losick, 2002;Kjos & Veening, 2014;Stracy et al, 2015;Woldringh, 2002). Transcription of many genes distributed throughout the genome might provide driving force for chromosome segregation, maintain appropriate chromosomal structure with supercoils separated by expressed regions, or reorganize the nucleoid within the cell (Kjos & Veening, 2014;Le et al, 2013;Stracy et al, 2015;Woldringh, 2002).…”