Fig. 4: Simultaneous biogenesis of all twenty aminoacyl-tRNA synthetases (aaRSs).
From: Autonomous biogenesis of all thirty proteins of the Escherichia coli translation machinery
a Schematic representation of aminoacylation: aaRS enzymes charge tRNAs with their cognate amino acids. b Difference in maximum GFP synthesis rates (Δkmax) at threshold with and without each aaRS gene plotted versus aaRS length (amino acids). Kendall’s rank correlation (see Methods) indicates a moderate negative correlation. Colors distinguish initial concentrations C0 = 0 and C0 > 0. Data are mean ± s.e.m., n = 3.c GFP FI(t) while expressing 18 aaRS genes (all except GlyRS and PheRS) in a bulk PURE reaction with three different threshold concentrations. Left: Cmin < 5 nM: AlaRS = 3.0, AspRS = 2.0, GlnRS = 0.8, IleRs = 0.5, LeuRS = 2.0, MetRs = 0.5, ProRs = 1, SerRS = 1, ValRs = 1. Middle and right: Cmin = 5 nM or 10 nM for all aaRS. [aaRS-DNA] = 0.1 nM (except for MetRS-DNA = 0.4 nM), [DNAGFP] = 0.4 nM (for replicates see Supplementary Fig. 21). d Scheme: DNA brush immobilized on a surface localizing the purified (dark blue) translation proteins and nascent (yellow) aaRS. e Prism-TIRF images of a hexagonal array of GFP-HA traps (green) and three DNA brushes (white dashed circles). Top: brush containing 2% GFP gene and 98% non-interacting dummy gene (DNA unlabeled). Bottom: 20% aaRS gene mix containing all 20 aaRS genes (Supplementary Table 3), 2% GFP gene, and 78% dummy genes; aaRS DNA is labeled (leftmost image is a red/green overlay). GFP-HA accumulates around brushes encoding aaRS genes. Scale bar, 200 µm. f Top: average GFP FI(t) from brushes with varying aaRS fractions (replicates in Supplementary Fig. 22). Bottom: mean fold change of GFP FI ± s.e.m from DNA brushes (normalized to the signal from “0%” brush) at t = 120 min ±s.e.m. as a function of total DNA FI (aaRS + GFP DNA) across brushes (n = 6 brush clusters from two slides). Source data are provided as a Source Data file.
