RNAP activity is reduced in 1N cells growing in M9glyCAAT.
(A) RpoC-YFP fluorescence concentration in 1N (3580 cells, CJW7477) and multi-N (5554 cells, CJW7563) cells as a function of cell area. Lines and shaded areas denote mean ± SD from three experiments. (B) Relative protein concentration of core RNAP subunits and σ70 in 1N-rich (SJ_XTL676) and multi-N (SJ_XTL229) cells by TMT-MS. 1N-rich cells were collected 0, 120, 180, 240, and 300 min after addition of 0.2% L-arabinose, while multi-N cells were collected after 0, 60, and 120 min of induction. (C) Apparent diffusion coefficients of JF549-labeled RpoC-HaloTag in WT (91,280 tracks from 1000 cells, CJW7519), 1N (175884 tracks from 1219 cells, CJW7520) and multi-N cells (186951 tracks from 1040 cells, CJW7527). Only tracks of length ≥9 displacements are included. 1N cells are binned according to cell area while multi-N cells contain aggregated data for ∼2-15 µm2 cell areas. (D) Da in WT cells fitted by a 3-state GMM: 49 ± 4%, 49 ± 4%, and 2 ± 0.1% (± SEM) of the RNAP population, from the slowest moving to the fastest moving (91,280 tracks from 1,000 cells). (E) Example WT and 1N cells where active (red, slow-moving) and inactive (gray, fast-moving) RNAPs are classified according to the GMM. (F) Active RNAP fraction in individual WT (854 cells) and 1N (1024 cells) cells as a function of cell area. Only cells with at least 50 tracks are included. Lines and shaded areas denote mean ± 95% CI of the mean from bootstrapping (3 experiments). (G) Same as (F) but for WT (854 cells) and multi-N (924 cells) cells. (H) Total amount of active RNAP in WT, 1N and multi-N cells as a function of cell area (calculated from Figure 3A, F, and G). Also, shown is a linear fit to multi-N data (f(x) = 4.16 · 104 · x, R2 0.98). Lines and shaded areas denote mean and 95% CI of the mean from bootstrapping. All microscopy data are from three biological replicates.