Single-step in vitro reconstitution of the Escherichia coli ribosome mediated by two GTPase factors, EngA and ObgE
- Laboratory for Cell-Free Protein Synthesis, RIKEN Center for Biosystems Dynamics Research (BDR), Japan
- Graduate School of Frontier Biosciences, The University of Osaka, Japan
- Laboratory of Nucleic Acid Nanotechnology, Institute for Quantitative Biosciences, The University of Tokyo, Japan
- Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Sweden
Figures
Figure 1
Ribosome assembly with two-step procedure.
(a) Schematic of the two-step procedure identified by Nierhaus and Dohme. In 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 step 2, increasing the incubation temperature and magnesium ion concentration is essential for the assembly of a translationally active 50S subunit. (b) Individual effect of temperature and magnesium concentration changes on the activity of assembled ribosomes.
Figure 2
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.
Figure 3
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 superfolder green fluorescent protein (sfGFP) fluorescence at 4 hr 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).
Figure 4
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 the help of ribosome biogenesis factors and then, assembled ribosomes exhibit the translation activity, which can be detected as superfolder green fluorescent protein (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 hr 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).
Figure 5
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 the coupled system is applied, magnesium concentration was fixed at 8 mM, and potassium concentration was fixed at 150 mM (a, c). When the 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 superfolder green fluorescent protein (sfGFP) fluorescence at the 8 hr timepoint, compared to that using all six factors (a, b), and the time course of sfGFP fluorescence (c) are shown.
Figure 6
Rate of increase in fluorescence of synthesized superfolder green fluorescent protein (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 represents the data of the assembled ribosome.
Figure 7
Characteristics of the synthesized proteins using the assembled ribosome.
(a) SDS-PAGE analysis of purified superfolder green fluorescent protein (sfGFP). sfGFP synthesis was performed with native ribosome (lane 1) and assembled ribosome (lanes 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 dihydrofolate reductase (DHFR). DHFR catalyzes the reduction of dihydrofolate acid into tetrahydrofolate acid, in which the reaction could be monitored through the reduction of 340 nm NADPH absorbance. Activities of synthesized DHFR with native ribosome and assembled ribosome were measured.
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Figure 7—source data 1
Original TIF file of the full raw, uncropped, and unedited SDS-PAGE gel image shown in Figure 7a.
- https://cdn.elifesciences.org/articles/109916/elife-109916-fig7-data1-v1.zip
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Figure 7—source data 2
PDF file containing the uncropped SDS-PAGE gel image shown in Figure 7a, with the relevant bands and lanes clearly labeled.
- https://cdn.elifesciences.org/articles/109916/elife-109916-fig7-data2-v1.zip
Figure 8
Reassembly of unfolded ribosomes using EngA and ObgE.
Ribosomes were unfolded by the addition of a high concentration of EDTA and then restored the environment by the addition of magnesium ions. Following this, reassembly of the ribosome was addressed by using EngA and ObgE. The time course of superfolder green fluorescent protein (sfGFP) fluorescence is shown in the presence or absence of EngA and ObgE.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/109916/elife-109916-mdarchecklist1-v1.pdf
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Source data 1
Mass spectrometry analysis of superfolder green fluorescent protein (sfGFP) synthesized by reconstituted ribosomes.
- https://cdn.elifesciences.org/articles/109916/elife-109916-data1-v1.xlsx
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Source data 2
Mass spectrometry analysis of TP70 used for ribosome reconstitution.
- https://cdn.elifesciences.org/articles/109916/elife-109916-data2-v1.xlsx
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Source data 3
DNA sequences of the plasmids used in this study.
- https://cdn.elifesciences.org/articles/109916/elife-109916-data3-v1.docx
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Single-step in vitro reconstitution of the Escherichia coli ribosome mediated by two GTPase factors, EngA and ObgE
eLife 15:RP109916.
https://doi.org/10.7554/eLife.109916.4
