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Expression of non‐open reading frames isolated from phage display due to translation reinitiation
Author(s) -
Song Liting,
Mandecki Wlodek,
Goldman Emanuel
Publication year - 2003
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.03-0105com
Subject(s) - open reading frame , start codon , upstream open reading frame , mutagenesis , translation (biology) , stop codon , genetics , biology , eukaryotic translation , reading frame , frameshift mutation , microbiology and biotechnology , mutation , peptide sequence , messenger rna , gene
An unusual 38 codon sequence was previously isolated from a random peptide library by binding to growth hormone binding protein in phage display. This sequence, “H10,” and several variants did not contain open reading frames, but expressed a β‐galactosidase reporter 10–40% as well as control in both the original reading frame from phage display and the frame −1 to it. Inspection of the sequence suggested that expression in the −1 frame resulted from initiation at a downstream ATG in that frame, present in H10 and its variants, subsequently confirmed by site‐directed mutagenesis. Unexpectedly, mutagenesis of that out‐of‐frame downstream ATG also increased expression in the original non‐open reading frame by two‐ to threefold, creating a TTG codon adjacent to an existing in‐frame TTG codon, suggesting downstream translational reinitiation at a putative TTG start. We undertook an extensive site‐directed mutagenesis approach and report that this hypothesis is almost certainly correct. Features required for this reinitiation include an upstream translation start and a stop that can even be a suppressed amber codon 22 nucleotides further downstream from the restart. Replacing the TTG with ATG increases expression only twofold. Reinitiation occurs in either of two reading frames in this sequence.—Song, L., Mandecki, W., Goldman, E. Expression of non‐open reading frames isolated from phage display due to translation reinitiation. FASEB J. 17, 1674–1681 (2003)