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. 2021 Sep 10;118(37):e2104577118. doi: 10.1073/pnas.2104577118

Fig. 4.

Fig. 4.

Translation suppression of Rtc1 in ΔDph2 is due to the presence of −1 frameshifting motifs. (A) A long frameshifting-prone sequence formed by three predicted frameshifting motifs in Rtc1. The −1 frameshifting yields two premature proteins with stop codon (TAG) highlighted in the sequence. The sizes of these premature proteins are labeled. (B) The scheme of the luciferase assay to test frameshifting efficiency. Frameshifting motifs are inserted between Renilla luciferase and Firefly luciferase. If frameshifting happens, the expression of Firefly luciferase will decrease. The ratio between the activity of Renilla luciferase and Firefly luciferase determines the frameshifting efficiency. (C) Two out of three frameshifting motifs in the long frameshifting-prone sequence show frameshifting-inducing effect. (D) Deletion of the frameshifting motif Rtc1(1836 to 1889) restores the translation efficiency of Rtc1(1 to 1998) in the reporter assay. (E) A premature Rtc1 was detected in ΔDph2 strain with Western blot upon the treatment of protease inhibitor, MG132. The Upper was the Flag signal of full-length Rtc1. The Lower was obtained by exposing the lower section of the same gel with Clarity Max ECL substrate (Bio-Rad). The size of the premature protein is in line with the −1 frameshifting in the long frameshifting-prone sequence of Rtc1. *P < 0.05; **P < 0.01; ***P < 0.001.