Boundary-Free Ribosome Compartmentalization by Gene Expression on a Surface

Abstract

The design of artificial cell models based on minimal surface-bound transcription-translation reactions aims to mimic the compartmentalization facilitated by organelles and inner interfaces in living cells. Dense DNA brushes as localized sources of RNA and proteins serve as synthetic operons that have recently proven useful for the autonomous synthesis and assembly of cellular machines. Here, we studied ribosome compartmentalization in a minimal gene-expression reaction on a surface in contact with a macroscopic reservoir. We first observed the accumulation and colocalization of RNA polymerases, ribosomes, nascent RNAs and proteins, in dense DNA brushes using evanescent field fluorescence, showing transcription-translation coupling in the brush. Fluorescence recovery after photobleaching showed that ribosomes engaged in translation in the brush had a 4-fold slower diffusion constant. In addition, ribosomes in the brush had over a 10-fold higher local concentration relative to free ribosomes, creating a boundary-free functional ribosome-rich compartment. To decouple translation from transcription, we immobilized dense phases of ribosomes next to DNA brushes. We demonstrated that immobilized ribosomes were capable of protein synthesis, forming 2D subcompartments of active ribosome patterns induced and regulated by DNA brush layout of coding and inhibitory genes. Localizing additional molecular components on the surface will further compartmentalize gene-expression reactions.

Figure 1

DNA brushes localize gene expression machinery and products. (A) Left: Scheme of TIRF microscopy setup. DNA brush immobilized on a biochip drives spontaneous recruitment of the gene expression machinery and gives rise to a protein source. Right: TIRF Imaging of the DNA brush end-labeled in red (647 nm). Scale bar: 100 μm. (B) Imaging of biomolecules colocalized with active coding brushes in independent experiments. T7 RNA polymerases, E. coli ribosomes and r-protein S2 are each fused to GFP (Methods). r-RNA is labeled with Broccoli aptamer. Free ribosomes are recruited to active DNA brushes but excluded from noncoding ones. The labeled species is depicted green above each image. (C) Signal kinetics of the labeled biomolecules measured in TIRF microscopy (independent experiments). Background was subtracted. Time t = 0 corresponds to the instance when the temperature crossed 30 °C in its rise from 17 to 37 °C. Error bars are standard deviation of 20–30 brushes.

Figure 2

Ribosome reduced mobility within DNA brushes. (A) Scheme of 2.5 kbp DNA brushes with a 400 bp gene located at the top, middle, and bottom of the brush. The promoter upstream of the gene is positioned 1956, 1262, and 687 bp from the tethered end of the DNA and its direction is depicted. The z position of ribosomes dictates their mobility and TIRF excitation. (B) TIRF signal for ribosomes localized on the three types of brushes as a function of brush density. Dashed lines are linear fits. Error bars are standard deviation of 3 brushes. (C) Scheme of FRAP experiment on labeled ribosomes (green) in a DNA brush at bleaching (gray) and during recovery. Recovery takes place through ribosome lateral mobility inside the brush and vertical exchange with ribosomes from solution. (D) TIRF images showing fluorescence recovery of labeled ribosomes on a NC brush and on a brush made of DNA from configuration 3 after photobleaching of an 80 μm circular region. Scale bars: 100 μm. (E) Normalized signal recovery after photobleaching at different time points of labeled ribosomes localized on brushes from configurations 1, 2, 3 as well as on NC DNA and free in solution. Solid lines are theoretical fits (Methods). (F) Diffusion coefficients averaged over three measurements in the first 30 min after beginning of gene expression, extracted from the theoretical fits (Methods). Error bars are standard deviation of the three measurements.

Figure 3

Localized protein sources. (A) Schemes, Top: A line of DNA brushes immobilized on a chip. Bottom: In each brush, transcription and translation colocalize and nascent proteins are trapped on patterned antibodies. (B) Top: Images of the TIRF signal that builds up symmetrically in time on two sides of a line of brushes coding for S15-GFP-HA. Bottom: Profiles of the signal. (C) Top: Scheme of lines of brushes, each with a different gene fraction φ, organized on a single surface 1800 μm apart, generating fluorescent signals of different intensities that were monitored in parallel. Bottom: Total fluorescent signal as a function of time for different gene density φ. Inset: Velocity of the front propagation as a function of gene density φ. (D) Scheme: r-RNA modified with Broccoli aptamer and r-protein S17-HA are synthesized and diffuse from nearby brushes. The binding of r-RNA to surface antibodies is mediated by S17-HA. (E) TIRF images of r-RNA signal buildup in time next to two lines of brushes, as in (D), with the r-protein S17-HA at gene fraction φ = 0.5, separated by 600 μm from the r-RNA brushes. The front of the signal propagates nonsymmetrically toward the right. Scale bar: 100 μm. (F) A space-time plot of the position of first appearance of the r-RNA signal (Y-axis) as a function of time (X-axis) for different distances d between lines of brushes, as defined in (E), with S17-HA brush gene fraction φ = 0.5 (blue) or φ = 0.05 (green). The r-RNA brushes have a fixed gene fraction φ = 0.5. The S17-HA brushes are positioned at the coordinates’ origin. The r-RNA brushes are positioned at a distance d, where each dotted line crosses the Y-axis.

Figure 4

Surface immobilized ribosome carpets. (A) Scheme (top) and TIRF image (bottom) of fluorescently labeled ribosomes immobilized on antibodies next to a DNA brush (dark circles exclude ribosomes) through the large ribosomal subunit (LSU). (B) Maximum GFP-SecM TIRF signal for different concentrations of small ribosomal subunit (SSU) in solution. Error bars are standard deviation of 12 regions. (C) Top: TIRF signal of GFP-SecM builds up symmetrically in time on surface ribosomes next to a line of coding brushes. Bottom: Signal profiles in time. (D) Top: Scheme of post-transcriptional regulation by asRNA hybridizing with GFP-SecM mRNA, inhibiting its translation on surface bound ribosomes. Bottom: TIRF images of maximal GFP-SecM (t = 34 min) in the two configurations schematized below. DNA brushes coding for GFP-SecM mRNA (red), asRNA (blue) or NC (black) are depicted as circles with unique arrangements. (E) Top: Scheme and TIRF image of GFP-SecM signal appearing on surface ribosomes around NC brushes but not around AS brushes, on the left and right of a line of GFP-SecM brushes, respectively. Bottom: Profiles of TIRF signal in time. Two configurations with reverse positions for the NC and AS brushes were averaged to avoid artifacts from flow. The positions of the NC (gray), GFP-SecM (red), and AS (brown) lines of brushes are marked. Scale bar: 100 μm.