Translation activity of chimeric ribosomes composed of Escherichia coli and Bacillus subtilis or Geobacillus stearothermophilus subunits

Abstract

Ribosome composition, consisting of rRNA and ribosomal proteins, is highly conserved among a broad range of organisms. However, biochemical studies focusing on ribosomal subunit exchangeability between organisms remain limited. In this study, we show that chimeric ribosomes, composed of Escherichia coli and Bacillus subtilis or E. coli and Geobacillus stearothermophilus subunits, are active for β-galactosidase translation in a highly purified E. coli translation system. Activities of the chimeric ribosomes showed only a modest decrease when using E. coli 30 S subunits, indicating functional conservation of the 50 S subunit between these bacterial species.

Keywords: Bacillus subtilis; Geobacillus stearothermophilus; In vitro translation; PURE system; Ribosome.

Fig. 1

Ribosome translation activity in a highly purifiedEscherichia colitranslation system. A) Time-course data of fluorescence as an indicator of E. coli 70S ribosome translation activity. Ribosomes were applied at the indicated concentrations to the β-galactosidase translation assay, as described in the Materials and Methods. Fluorescence produced by the translated β-galactosidase was monitored every 10 min over a total of 15 h. The experiments were performed in triplicate for each ribosome concentration. B) The maximum slope in 1A was plotted as an index of translation activity. The control experiment without lacZ DNA was also performed (- lacZ). The error bars indicate standard deviation (n=3).

Fig. 2

Translation activity ofEscherichia coliandBacillus subtilischimeric ribosomes.E. coli (Ec) and B. subtilis (Bs) ribosomal subunits (30 nM) were prepared separately and mixed in the highly purified E. coli translation system at the indicated 30S and 50S combinations. The translation activity of each chimeric ribosome was monitored by fluorescence produced by translated β-galactosidase. As an index of translation activity, the maximum rate of fluorescence increase is shown for 10 h reaction time. The error bars indicate standard deviation (n=3). P-values between lanes 1 and 5, 1 and 6, 3 and 4, 3 and 5, and 3 and 8 are <0.03, <0.03, <0.001, <0.001, <0.001, respectively.

Fig. 3

Translation activity ofEscherichia coliandGeobacillus stearothermophiluschimeric ribosomes.E. coli (Ec) and G. stearothermophilus (Gs) ribosomal subunits (100 nM) were prepared separately and mixed in the highly purified E. coli translation system at the indicated 30 S and 50 S combinations. The translation activity of each chimeric ribosome was monitored by fluorescence produced by translated β-galactosidase. As an index of translation activity, the maximum rate of fluorescence increase is shown for 10 h reaction time. The error bars indicate standard deviation (n=3). P-values between lanes 1 and 5, 1 and 6, 2 and 7, 2 and 8, 3 and 4, 3 and 5, and 3 and 8 are <0.0001, <0.0001, <0.005, <0.005, <0.001, <0.005, <0.005, <0.01, <0.01, respectively.