Fig. 4: LSU biogenesis in vitro.
A Experimental scheme for LSU biogenesis in vitro. B Exploring optimal conditions for the first reaction. In the first reaction, the concentrations of the native ribosomes, artificial rRNA operon with A2058U clindamycin resistance (CldR), and 33 LSU r-protein genes were 80, 0.6–1.5, and 0–0.015 nM each, respectively. The second reaction was conducted with the WT-SD–LacZ reporter and clindamycin using the bulk assay. a.u., arbitrary unit. C Reproducible detection of the nascent artificial LSU translational activity under the optimized reaction condition. In the first reaction, the concentrations of the native ribosomes, the artificial rRNA operon, and 33 LSU r-protein genes were 80, 0.9, and 0.01 nM each, respectively. The second reaction was conducted with the WT-SD–LacZ reporter and clindamycin using the bulk assay. Mean ± SD (n = 3, biological replicates). ****, p < 0.0001; n.s., not significant; one-way ANOVA with Dunnett’s test against the negative control without native ribosomes. The p-value showing p  <  0.0001 is p  =  1.0 × 10−5. D Improvement of the nascent LSU-derived fluorescence signal. The experimental condition was the same as Fig. 4C except that an improved LacZ reporter with a modified 5′UTR sequence was used instead of the WT-SD–LacZ reporter. The signal-to-noise ratio was improved because the background translation by the native ribosomes was sufficiently suppressed by 1.5 mM clindamycin (Supplementary Fig. 7A). Mean ± SD (n = 3, biological replicates). *, p < 0.05; two-tailed Welch’s t-test. Although the experimental conditions in lane 1 in Fig. 4C and lane 3 in Fig. 4D were identical, they showed different values because these experiments were performed on different days. Source data are provided as a Source Data file.
