Low copy number plasmids in bacteria require segregation for stable inheritance through cell division. This is often achieved by a parABC locus, comprising an ATPase ParA, DNA-binding protein ParB and a parC region, encoding ParB-binding sites. These minimal components space plasmids equally over the nucleoid, yet the underlying mechanism is not understood. Here we investigate a model where ParA-ATP can dynamically associate to the nucleoid and is hydrolyzed by plasmid-associated ParB, thereby creating nucleoid-bound, self-organizing ParA concentration gradients. We show mathematically that differences between competing ParA concentrations on either side of a plasmid can specify regular plasmid positioning. Such positioning can be achieved regardless of the exact mechanism of plasmid movement, including plasmid diffusion with ParA-mediated immobilization or directed plasmid motion induced by ParB/parC-stimulated ParA structure disassembly. However, we find experimentally that parABC from Escherichia coli plasmid pB171 increases plasmid mobility, inconsistent with diffusion/immobilization. Instead our observations favor directed plasmid motion. Our model predicts less oscillatory ParA dynamics than previously believed, a prediction we verify experimentally. We also show that ParA localization and plasmid positioning depend on the underlying nucleoid morphology, indicating that the chromosomal architecture constrains ParA structure formation. Our directed motion model unifies previously contradictory models for plasmid segregation and provides a robust mechanistic basis for self-organized plasmid spacing that may be widely applicable.

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The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼25-fold with elongation rate varying ∼8-to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context.he influence of chromatin on transcription and cotranscriptional processing is of central importance in the regulation of gene expression (1, 2). An intensively studied example where the local chromatin state is considered to influence transcription in Arabidopsis is FLOWERING LOCUS C (FLC). FLC encodes a MADS-box transcription factor and acts as a floral repressor (3, 4). FLC expression is tuned by different genetic pathways: FRIGIDA activates FLC expression through a mechanism requiring Trithorax homologs, Paf1C, and SET DOMAIN GROUP 8 (SDG8), an H3K36 methyltransferase (5). FLC expression is repressed by the autonomous pathway and vernalization (5). Both these repressive pathways involve a group of antisense long noncoding transcripts collectively termed COOLAIR, which initiate immediately downstream of the poly(A) site at the 3′ end of FLC. These antisense transcripts terminate at either proximal sites internal to the FLC gene, or distal sites within the FLC promoter (6, 7). Mutation of autonomous pathway components, including the RNA binding proteins FCA and FPA and the conserved components of the 3′ processing complex FY, Cstf64 and Cstf77, leads to relative reduction in use of the proximal polyadenylation sites and increased FLC sense expression (reviewed in ref. 8). FCA localizes to FLC chromatin near the proximal poly(A) sites (9), and this together with the fact that PRP8 and CDKC;2 (P-TEFb component), identified in FCA suppressor screens (10, 11), both require COOLAIR to repress FLC, supports the idea that promotion of proximal polyadenylation of COOLAIR is directly linked to reduced FLC expression. FLOWERING LOCUS D (FLD), an H3K4me2 demethylase, als...

SummarySingle-cell quantification of transcription kinetics and variability promotes a mechanistic understanding of gene regulation. Here, using single-molecule RNA fluorescence in situ hybridization and mathematical modeling, we dissect cellular RNA dynamics for Arabidopsis FLOWERING LOCUS C (FLC). FLC expression quantitatively determines flowering time and is regulated by antisense (COOLAIR) transcription. In cells without observable COOLAIR expression, we quantify FLC transcription initiation, elongation, intron processing, and lariat degradation, as well as mRNA release from the locus and degradation. In these heterogeneously sized cells, FLC mRNA number increases linearly with cell size, resulting in a large cell-to-cell variability in transcript level. This variation is accounted for by cell-size-dependent, Poissonian FLC mRNA production, but not by large transcriptional bursts. In COOLAIR-expressing cells, however, antisense transcription increases with cell size and contributes to FLC transcription decreasing with cell size. Our analysis therefore reveals an unexpected role for antisense transcription in modulating the scaling of transcription with cell size.