Autonomous synthesis and assembly of a ribosomal subunit on a chip

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Science Advances  15 Apr 2020:
Vol. 6, no. 16, eaaz6020
DOI: 10.1126/sciadv.aaz6020
  • Fig. 1 A genetic program for ribosome assembly on a chip.

    (A) Scheme: r-RNA (in situ fluorescently labeled; green spot), r-proteins, and assembly factors expressed from gene brushes assemble and bind to surface anti-HA antibodies through an HA tag on one of the r-proteins (orange triangle). (B) A radial brush layout (top scheme) of r-RNA (green) and r-protein–HA (black) genes in the central three-brush cluster, surrounded by gene brushes of six assembly factors (grayscale) and all other r-proteins (RGB-scale), related by color to the E. coli assembly map [bottom scheme; based on (4) and (18)]. r-proteins in the same brush are shaded green, classified as primary, secondary, or tertiary, and as 5′, central or 3′ domains of the r-RNA (green line). Arrows indicate the dependency order. (C) Top: Twenty three-brush clusters on the same surface, each with a different r-protein–HA gene, produce variable r-RNA signals. Bottom: Fluorescent images of r-RNA binding to S8-HA at four time points. Scale bar, 100 μm. (D) r-RNA can bind to r-protein–HA on the surface or in solution before surface binding. (E) Signal dynamics of r-RNA interaction with primary (P) (left), secondary (S) (center), and tertiary (T) (right) r-protein–HAs in the absence of assembly factors. Bottom: fmax of each primary, secondary, and tertiary r-protein–HAs. Error bars are standard deviation (SD) of three repeats. Time intervals are averages of three repeats. a.u., arbitrary units.

  • Fig. 2 E. coli SSU biogenesis on a chip.

    (A) Scheme: Twenty brush clusters coding for all SSU r-proteins, and the assembly factors Era, RsgA, RbfA, RimM, RimN, and RimP surround the central r-RNA and r-protein-HA brushes. (B) r-RNA fluorescent signal buildup in time for the S15-HA configuration. Scale bar, 100 μm. (C) Top: Signal dynamics of r-RNA binding to primary (left), secondary (center), and tertiary (right) r-proteins–HAs. Bottom: fmax of all r-proteins–HAs. (D) SSU t0 timeline indicating the onset time of each r-RNA:r-protein–HA interaction. Error bars are SD of three repeats. Time intervals are averages of three repeats.

  • Fig. 3 Binding dependencies of E. coli secondary r-proteins.

    (A) Scheme: Two modes of r-RNA binding to r-protein–HA on the surface, dependent on prebinding of other r-proteins to the r-RNA, in the absence of assembly factors. (B) Brush layouts (a1 to a8) of central domain analysis (S6-HA) and the corresponding fluorescent images at t = 45 min. Scale bar, 100 μm. (C to E) Signal dynamics color maps of brush combinations, central (a1 to a15), 5′ (b1 to b8), and 3′ (c1 to c8, d1 to d8, e1 to e8) domains. White time intervals represent t0. Gene combinations are depicted as gray and white boxes, indicating the presence or absence of an r-protein gene, respectively. Red frame indicates the r-protein–HA. Averaged fmax values are presented as bars at the end of each color map and as a Venn diagram according to the color scale. Error bars are SD of three repeats. Partial assembly maps depict in red the dependencies deduced from the corresponding study. Large arrows represent strong dependencies.

  • Fig. 4 Late stages of on-chip E. coli SSU assembly.

    (A) r-RNA:S2-HA signals for different combinations of SSU domains (a1 to a8), depicted as dynamic color maps. Labels are as in Fig. 3. (B) Averaged fmax values of r-RNA:S2-HA signals for different assembly factor combinations with and without all r-proteins. Error bars represent SD of three repeats. Scale bar, 100 μm. (C) Signal dynamics of r-RNA:S2-HA interactions in the presence of four factors and domain combinations. (D) Scheme: Ribosomes localized on surface antibodies actively engaged in GFP-SecM synthesis with purified SSU added from bulk solution. mRNA is produced from nearby DNA brushes. Inset: TIRF dynamic signal of GFP-secM on surface-bound ribosomes. (E) Scheme: Synthesis and assembly of nascent SSU and binding to surface immobilized LSUs. (F) Signal dynamics color maps of nascent SSU binding onto surface-bound LSU, dependent on different r-protein combinations (b1 to b4). In each panel, the presence of assembly factors is marked as 2F (Era and RsgA), 4F (RbfA, RimM, RimN, and RimP), or 6F (all).

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