Fig. 3: The rate of transcriptional elongation and of protein synthesis on a single chromosome.
From: Bacterial chromosome conformation and cell-free gene expression in synthetic 2D compartments
a Fluorescence time-lapse images of an E. coli chromosome in a compartment labeled by nucleoid-associated proteins (HUα-GFP, cyan) and RNAP (β’-HT, magenta). At t = 0 min, the lysis buffer was exchanged for a TxTl system. The decay of the signal is shown overlayed (left) and in separate (right) channels. b Decay of fluorescence signals of HUα-GFP and β’-HT from one chromosome upon introducing the TxTl system, reporting on the rate of transcriptional elongation, followed by decay and dilution out of the compartment. The dynamics were observed for three chromosomes. c Fluorescence signals of RNAP (β’-HT) upon introducing the TxTl system as in (b) for three conditions: active transcription (not blocked) (black curve), blocked transcription initiation by rifampicin (Rif, yellow curve), and depleted of energy buffer for transcription (red curve). The dynamics were observed with five chromosomes in at least two independent biological experiments for each condition. d Schematics for measuring the protein synthesis rate from a chromosome engineered with a HaloTag (HT) gene cassette. Nascent HA-tagged HaloTag proteins were captured through surface-immobilized Anti-HA antibodies for imaging. e Exemplary dual-color fluorescence image of a chromosome labeled with HUα-GFP, and two surface-captured HA-tagged HaloTag proteins (white arrows). f Time traces of arrival time and counts of HA-HaloTag protein spots captured and recorded on the surface during the TxTl reaction from a chromosome in the compartment. g The rate of HaloTag proteins expressed in a TxTl reaction with single plasmids, chromosomes, and without DNA (as a negative control). Data show independent replicates, and the bars with error bars (S.D.) represent their means. Two-sided Mann-Whitney U tests were performed to compute the statistical significance. The cumulative distributions of protein arrival times are shown with fits to mono-exponential distributions (black lines). The fits gave arrival rates of 0.67 ± 0.05 min−1 for chromosomes and 0.37 ± 0.01 min−1 for plasmids. The data were combined from independent experiments with plasmids (N = 7) and chromosomes (N = 3). Compartment diameter, 20 µm. The dashed white lines outline the compartments and capillaries connecting to the two main flow channels. Source data is provided as a Source Data file.
