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. 2020 Jun 22;117(27):15609–15619. doi: 10.1073/pnas.1914323117

Fig. 5.

Fig. 5.

Characterization of the ∆rrmJ ribosomes lacking U2552 methylation in fast kinetics-based translation assays. All data are fitted using single or double exponential functions (as indicated) and the rates are estimated using Origin 8.0. All experiments were done at least three times. The rates are presented with SDs. (A) Kinetics of association of the 30S preIC with the 50S subunits from the WT (black square) and the ΔrrmJ (red circle) cells. The formation of the 70S IC is followed by increase in Rayleigh light scattering at 365 nm in a stopped-flow instrument. The curves are fitted with double exponential function and the rates are estimated from the fast phase. (B) Kinetics of fMet-Phe dipeptide formation in quench flow from 70S IC from WT (black square) and ΔrrmJ (red circle) cells under the single-turnover condition. The curves are fitted with a single exponential function to obtain the reaction rates. (C and D) Kinetics of fMet-Phe-Leu tripeptide formation from 70S IC (WT,black square and ΔrrmJ, red circle) under single-turnover (C) and multiple-turnover (D) conditions, done in quench flow (C) and manual mixing (D), respectively. The curves are fitted with single exponential function and the rates are estimated. (E and F) Kinetics of EF-G mediated translocation with the ribosomes from WT (black square) and ΔrrmJ (red circle) cells under single-turnover (E) and multiple-turnover (F) conditions. The 3′ pyrene labeled MFL mRNA was used in this assay, where decrease in the pyrene fluorescence representing mRNA movement upon translocation is monitored in stopped-flow (excitaton 343 nm and emission 360 nm). The curves are fitted with single exponential function and the rates are estimated. (G) Kinetics of BOP-Met release from the RC programmed with Met-Stop (UAA) mRNA by RF1 (WT, black square and ΔrrmJ, red circle) and RF2 (WT, blue triangle and ΔrrmJ, green triangle). The rates are estimated from the fast phase by fitting the curves with double exponential function. (H) Kinetics of RRF and EF-G mediated splitting of the posttermination ribosomal complex from the WT (black square) and the ΔrrmJ (red circle) cells, followed by monitoring decrease in Rayleigh light scattering at 365 nm in a stopped-flow instrument. The curves are fitted with single exponential function and the rates are estimated.