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. 2016 Dec 9;45(3):1307–1318. doi: 10.1093/nar/gkw1239

Figure 3.

Figure 3.

DDX19B increases TC formation. (A) A scheme of toe-print analysis of a ribosome–mRNA complex formation. Stable ribosomal complex on the mRNA stops reverse transcriptase (AMV) at a certain position generating cDNA products of specific lengths. cDNA molecules synthesized with fluorescently labeled primers are separated and detected using fragment analysis. (B) Examples of raw data from capillary electrophoresis of cDNA products obtained using fluorescently labeled primers for toe-print analysis. PreTCs are shown after SDG purification, TC formation is induced by addition of eRF1 and eRF3c to the preTCs. Positions of preTCs and TCs are labeled by white and black triangles, respectively. (C) Toe-print analysis of termination complexes formed by addition to the preTCs of eRF1, eRF1+eRF3c+GTP, eRF1+eRF3a+GTP and DDX19B+ATP. Rfu – relative fluorescence unit. Positions of preTCs and TCs are labeled by white and black triangles, respectively. (D) Relative quantitative analysis of the stop codon binding efficiency of eRFs in the presence of DDX19B. Stop codon binding efficiency of eRF1 alone was set as 100%. The error bars represent the standard deviation, stars (**) mark a significant difference from the respective control P < 0.01 (n = 3).