Skip to main content
. 2015 Mar 25;10(3):e0122017. doi: 10.1371/journal.pone.0122017

Fig 4. Lifetimes of the translation arrest of HaloTag proteins harbouring the arrest sequence.

Fig 4

(A-D) Time-course analyses of polypeptidyl-tRNA remaining after the addition of puromycin. Halo-L17-SecM133–170 (A), Halo-L26-SecM133–170 (B), Halo-pD-L8-SecM133–170 (C) and Halo-SecM1–170 (D) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the mixture was incubated at 37°C. Aliquots removed at the indicated time points were subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. (E) Plots of the fraction of polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Squares, Halo-L17-SecM133–170; diamonds, Halo-L26-SecM133–170; triangles, Halo-pD-L8-SecM133–170; circles, Halo-SecM1–170. Data points represent means ± SD of three independent experiments. The solid and dotted lines show the fit to the data obtained using a single exponential function. The lifetimes of the translation arrest of Halo-L17-SecM133–170, Halo-L26-SecM133–170, Halo-pD-L8-SecM133–170 and Halo-SecM1–170 were 5.6 ± 0.066, 11 ± 0.22, 9.4 ± 0.63 and 51 ± 1.6 min, respectively (the errors represent fitting errors). (F) Time-course analysis of myc-SecM1–170 polypeptidyl-tRNA remaining after the addition of puromycin. Myc-SecM1–170 was translated using the PURExpress ΔRibosome Kit at 37°C for 40 min. Puromycin (1 mg/mL) was added at 0 min, and the mixture was incubated at 37°C. Aliquots were withdrawn at indicated time points and subjected to NuPAGE. Myc-SecM1–170 was detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. (G) The fraction of myc-SecM1–170 polypeptidyl-tRNA remaining in the presence of puromycin as a function of time. Data points with error bars represent means ± SD for three independent experiments. The solid line shows the fit to the data obtained using a single exponential function. The lifetime of the translation arrest of myc-SecM1–170 was 48 min ± 4.3 min (the error corresponds to fitting error).