Transcription-directed membrane association organizes the chloroplast nucleoid structure

ABSTRACT DNA is organized into chromatin-like structures, which support the maintenance and regulation of genomes. A unique and poorly understood form of DNA packaging exists in chloroplasts, which are endosymbiotic organelles responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membraneless structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA packaging, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and higher DNA accessibility. Disruption of transcription at specific genes in sigma factor mutants causes a corresponding reduction in membrane association, indicating that RNA polymerase activity causes DNA tethering to the membranes. We propose that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.