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. 2015 Jun 3;43(13):6426–6438. doi: 10.1093/nar/gkv558

Figure 3.

Figure 3.

Effect of RF2 on Sec insertion in vivo. (A) Co-expression of Fluc–FdhF–Rluc and RF2 constructs. prf(SPF) is a construct for 0-frame expression of RF2 with the native stop-codon recognition motif (SPF). In the prf(APA) construct, the SPF motif is mutated to APA to produce RF2 that is inactive in translation termination. (B) Example of a sodium dodecyl sulphate-polyacrylamide gel electrophoresis separation of the Fluc–FdhF–Rluc expression products in the presence of RF2 (SPF or APA) obtained from a single-copy (SC) or multi-copy (MC) plasmids. Fluc-FdhF and Fluc–FdhF–Rluc products were visualized by western blot analysis using an anti-Fluc antibody. The identity of the smaller peptides observed with the UUC+SECIS construct is not known. The amounts of RF2 in the same samples were detected using an antibody against the His-tag of RF2 (see also Supplementary Figure S1). (C) UGA recoding analysis from the western blot band intensity. Recoding was calculated as the ratio of the read-through band over the sum of the read-through and truncated bands. Error bars represent SEM from two experiments. The extent of read-through obtained with the UUC+SECIS construct defines 100% read-through efficiency. (D) Direct comparison of the read-through band intensities for all constructs. The band intensity for the product of UUC+SECIS translation at the SC RF2 conditions was taken as 1. Error bars represent SEM from two independent experiments.