Fig. 3.

The NΔ TBCE HRD allele is expressed as a result of translational initiation at three upstream out-of-frame AUG codons. (A) Nucleotide and corresponding amino acid sequences of wild-type TBCE (Upper) and the allele with the HRD deletion of nucleotides 66–67 (Lower) in the vicinity of the 2-bp deletion. Deleted nucleotides in HRD are shown (Upper) in red. Potential translational reading frames in the mutant HRD allele are shaded in gray and yellow. Numbering of amino acids in the −2 reading frame (1′, 2′, etc.) starts at the first residue (Q) of this open frame preceding the 2-bp deletion. Three M residues in this reading frame are highlighted in turquoise. Note that translational initiation at these methionines in the NΔ HRD allele leads to a transition to translation from the correct ORF, whereas translation initiated at the “correct” methionine in the mutant sequence (amino acids highlighted in brown) leads to termination at a stop codon located at nucleotides 148–150 (not shown). A polypeptide (MENMQLR) identified by mass spectroscopy in a tryptic digest of the protein expressed from the mutant HRD allele (see Fig. 6) is boxed. (B) Translational analysis of the NΔ HRD allele by site-directed mutagenesis. The mutated forms shown (corresponding triplets altered to alanine or termination codons are highlighted in A in pink and blue, respectively) were expressed as ³⁵S-labeled proteins by coupled transcription/translation in rabbit reticulocyte lysate and the reaction products detected by autoradiography following resolution by SDS/PAGE.