Ribosome assembly with two-step procedure.

(a) Schematic of the two-step procedure identified by Nierhaus and Dohme. In the step 1, comparatively low magnesium ion concentration and low incubation temperature are required to assemble the intermediate state of the 50S subunit (41S/48S particle), whereas in the step 2, increasing the incubation temperature and magnesium ion concentration are essential for the assembly of translationally active 50S subunit. (b) Individual effect of temperature and magnesium concentration changes on the activity of assembled ribosomes.

Ribosome assembly with ribosome biogenesis factors.

(a) Working hypothesis of this study. We hypothesized that some of the ribosome biogenesis factors take over the role of temperature and magnesium concentration changes in the two-step procedure. We selected six GTPase factors as candidates of these factors. (b) Single-step ribosome assembly by selected ribosome biogenesis factors. Activity of assembled ribosomes in the presence or absence of six factors at 8 or 17 mM magnesium ion concentration at 37 °C. Concentration of potassium glutamate for the ribosome assembly experiment was fixed at 200 mM.

Effect of magnesium and potassium concentrations on the single-step ribosome assembly using six GTPase factors.

(a) Effect of magnesium concentration on the assembled ribosome activity. (b) Effect of potassium concentration on the assembled ribosome activity. Relative endpoint sfGFP fluorescence at 4 h timepoint, compared to 8 mM magnesium or 300 mM potassium, respectively, is shown. Potassium concentration was fixed at 200 mM (a) and magnesium concentration was fixed at 8 mM (b).

Coupling of the assembly and translation reactions.

(a) Schematic of the coupled reaction. Three rRNAs (5S, 16S, and 23S) and TP70 are added into the ribosome-free PURE system. Ribosomes are assembled in the PURE system with a help of ribosome biogenesis factors and then, assembled ribosomes exhibit the translation activity, which can be detected as sfGFP fluorescence. (b) Time course of sfGFP fluorescence in the coupled reaction system. Effect of magnesium concentration is shown. (c) Effect of magnesium concentration on the assembled ribosome activity. (d) Effect of potassium concentration on the assembled ribosome activity. Relative endpoint sfGFP fluorescence at 8 h timepoint, compared to 8 mM magnesium or 150 mM potassium, respectively, is shown. Potassium concentration was fixed at 150 mM (b, c) and magnesium concentration was fixed at 8 mM (d).

Identification of the responsible factors.

(a) Ribosome activities assembled in the absence of each factor in the coupled system. (b) Ribosome activities assembled in the absence of each factor in the uncoupled system. (c) Ribosome activities assembled with only EngA and ObgE. When coupled system is applied, magnesium concentration was fixed at 8 mM and potassium concentration was fixed at 150 mM (a, c). When uncoupled system was used, magnesium concentration was fixed at 8 mM and potassium concentration was fixed at 250 mM for the ribosome assembly reaction (b). Relative endpoint sfGFP fluorescence at 8 h timepoint, compared to that using all six factors (a, b) or time course of sfGFP fluorescence (c) are shown.

Rate of increase in fluorescence of synthesized sfGFP.

(a) sfGFP synthesis was measured in the coupled system with varying concentrations of native ribosome. (b) The estimated concentration of assembled ribosomes was calculated from the native ribosome data. Black dots represent data of the native ribosome and a white dot represent the data of the assembled ribosome.

Characterisitics of the synthesized proteins using the assembled ribosome.

(a) SDS-PAGE analysis of purified sfGFP. sfGFP synthesis was performed with native ribosome (lane 1) and assembled ribosome (lane 2-4). sfGFP was not synthesized in the absence of EngA and ObgE (lane 3) and in the absence of sfGFP gene (lane 4). M represents the protein size markers. (b) Mass spectrometry analysis of the purified sfGFP. Amino acid sequence of the synthesized sfGFP is shown and the MS-detected sequences are marked with yellow. (c) Activity measurement of the synthesized DHFR. DHFR catalyzes reduction of dihydrofolate acid into tetrahydrofolate acid, in which the reaction could be monitored through reduction of 340 nm NADPH absorbance. Activities of synthesized DHFR with native ribosome and assembled ribosome was measured.

Reassembly of unfolded ribosomes using EngA and ObgE.

Ribosomes were unfolded by the addition of high concentration of EDTA and then restored the environment by the addition of magnesium ions. Followingly, reassembly of the ribosome was addressed by using EngA and ObgE. Time course of sfGFP fluorescence are shown in the presence or absence of EngA and ObgE..