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. 2017 May 24;12(5):e0177972. doi: 10.1371/journal.pone.0177972

Fig 7. Lack of IF3 effects during disassembly of the PoTC by RRF and EF-G.

Fig 7

(A) The release of mRNA and splitting were measured as described in Materials and Methods. When IF3 was present, the final concentration was 4.5 μM. The reaction was performed in buffer R2. Y axis for mRNA release represents the percentage of lost area of PoTC (polysome area) after the reaction. Y axis for splitting indicates the percentage of 50S area compared to the total area of PoTC at zero time. The areas were measured using imageJ (NIH). Depending on the preparation of PoTC, the activity varies. (B) IF3 alone does not split the ribosome into subunits: 0.05 μM of the PoTC, 5 μM of EF-G, 500 μM of GTP and increasing concentrations IF3 (as indicated in the figure) were mixed in the presence or absence of 5 μM of RRF in buffer R2 and incubated for 3 minutes at 30°C. Final volume of the reaction was 60 μL. The reaction was stopped with 100 μL of ice cold buffer R1 and loaded on a sucrose gradient of 15–35% in buffer R1. The gradients were spun for 3 hours and 30 min using an SW50 rotor at 40,000 RPM and analyzed using a ISCO UA-6 spectrophotometer at 254 nm. Sedimentation was from left to right. The sedimentation positions of the 50S, 30S and 70S ribosomal particles are indicated in the figure. IF3 alone was unable to split the PoTC into subunits even at high concentration (20 μM), whereas when RRF was present in the reaction mixture, the 50S and 30S subunits were easily detectable, indicating the splitting of the PoTC. (C) IF3 is not needed for tRNA release. The release of tRNA was measured as described in Materials and Methods. When IF3 was present, the final concentration was 4.5 μM. (D) IF3 does not stimulate the initial rate of ribosome splitting but is required for preventing the split subunits from re-associating. Splitting of ribosome was measured as described in Materials and Methods. When present, IF3 was 4.5 μM. The sedimentation pattern as shown in S4 Fig was obtained, and the area of the 50S peak was measured and plotted against incubation time.