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Results: 5

1.
Figure 5.

Figure 5. From: Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components.

Testing the functional conservation of resurrected ancient elongation factors (indicated at the bottom) in the reconstituted T. thermophilus system in which Tt Tu is replaced by each of the ancient elongation factors (indicated at the bottom) (Supplementary Figure S5). The translation reactions containing stGFP mRNA are conducted at 50°C for 4 h. The relative synthesis yields (%) are based on the fluorescence of the reactions compared with that of the original reconstituted system (Tt Tu, set as 100). All data are based on at least two independent experiments.

Ying Zhou, et al. Nucleic Acids Res. 2012 September;40(16):7932-7945.
2.
Figure 3.

Figure 3. From: Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components.

Contribution of TFs and ER to the overall protein synthesis in the reconstituted T. thermophilus system. The translation of stGFP mRNA is conducted at 50°C for 4 h in the reconstituted T. thermophilus system in which one or several TFs or ER are removed. The relative synthesis yields (%) are based on the fluorescence of the reactions compared with that of the complete reconstituted system (Complete, set as 100). ΔmRNA: missing stGFP mRNA; ΔTFs: missing all TFs; ΔIFs: missing all three initiation factors; ΔER: missing all ER; also see Supplementary Table S1 for the abbreviations. The data are based on at least two independent experiments.

Ying Zhou, et al. Nucleic Acids Res. 2012 September;40(16):7932-7945.
3.
Figure 4.

Figure 4. From: Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components.

Testing the functional compatibility of translation components between T. thermophilus and E. coli. (A) The exchange of key translation components (IFs, Tu/Ts, G, RRF and ribosome) between T. thermophilus (filled circles) and E. coli (open circles) in the reconstituted T. thermophilus system translating stGFP mRNA at 37°C. The relevant exchange experiments including the controls are boxed for clarity. (B) The exchange of EF-Tu and EF-Ts (Tu and Ts) between T. thermophilus (filled circles) and E. coli (open circles) in the reconstituted E. coli system translating stGFP mRNA at 37°C. The missing components are indicated by the minus symbol (−). The relative synthesis yields (%) are based on the fluorescence of the reactions compared with that of the original reconstituted systems (set as 100; lane 11 in A and lane 9 in B). All data are based on at least two independent experiments.

Ying Zhou, et al. Nucleic Acids Res. 2012 September;40(16):7932-7945.
4.
Figure 1.

Figure 1. From: Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components.

(A) Real-time monitoring of the fluorescence of stGFP synthesized in the reconstituted T. thermophilus system at 37°C (grey solid line: with stGFP mRNA and grey dashed line: without mRNA) and 50°C (black solid line: with stGFP mRNA and black dashed line: without mRNA). Inset: western blot analysis of aliquots taken from the 50°C reaction containing stGFP mRNA at different time points using an antibody specific to stGFP. (B) SDS–PAGE analysis of aliquots of the translation reactions at 4 h in the presence (lane 6) or absence (lane 5) of stGFP mRNA. Different amounts of purified recombinant stGFP are loaded on the same gel in lane 1 (200 μg/ml), lane 2 (100 μg/ml) and lane 3 (50 μg/ml). The MW standards (run in lane 4) are indicated on the left.

Ying Zhou, et al. Nucleic Acids Res. 2012 September;40(16):7932-7945.
5.
Figure 2.

Figure 2. From: Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components.

(A) Detection of the synthesis of target proteins (indicated at the bottom of lane 2–7) with [35S] methionine labeling in the reconstituted T. thermophilus system. As a control, mRNA is not added (lane 1, mRNA). BstYI (23 kDa) and PspGI (32 kDa): restriction enzymes from Bacillus stearothermophilus and Pyroccocus species, respectively; TAP (57 kDa): an alkaline phosphatase from T. thermophilus; AMase: (59 kDa): an amylomaltase from T. thermophilus; Vent DNAP (90 kDa): a DNA polymerase from hyperthermophilc Thermocuccus litoralis; stGFP (29 kDa): an engineered thermostable GFP variant from this study. The MW standards (MW, kDa) are indicated on the left. (B) Synthesis of Vent DNAP with [35S] methionine labeling at different temperatures in the reconstituted T. thermophilus system. (C) Comparison of the stability of mRNA at 60°C in in vitro translation reactions of the reconstituted T. thermophilus system (left panel) or T. thermophilus cell extract (right panel). The stability of 32P-labeled BstYI mRNA at various time points (indicated on top) is analyzed on a SDS-PAGE gel, followed by autoradiography. Purified 32P-labeled BstYI mRNA without incubation is shown as the control (ctrl). The RNA standards in base pairs (bp) are indicated on the left.

Ying Zhou, et al. Nucleic Acids Res. 2012 September;40(16):7932-7945.

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