Figure 1.
Characterization of CFTR TM8 read-through polypeptides. (A) Diagram of putative structure and topology of CFTR. Closed circles show location of two N-linked glycosylation sites in ECL4. Arrows represent approximate location of CFTR truncation sites in ECL4 (aa 935, 957, 967, 977, and 987), in TM9 (aa 997 and 1007), and in TM10 (aa 1017 and 1027). Methionine residue 837 initiates translation of TM7-8 construct. (B) TM8 sequence showing location of UAG sites (912, 913, and 914). Putative boundaries of CFTR TM8 are shown. (C) In vitro translation of truncated mRNAs in the presence and absence of Lys-tRNACUA (even- and odd-numbered lanes, respectively). Polypeptides that terminated at the amber codon (nRT) were partially glycosylated at two N-linked consensus sites (downward arrows). Read-through (RT) of the UAG codon generated larger polypeptides (asterisks) that were also singly or doubly glycosylated (g and gg, respectively). (D) As in C, except that glycosylation was suppressed by addition of the tripeptide NYT during translation.