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Expression of non-open reading frames isolated from phage display due to translation reinitiation

Department of Microbiology & Molecular Genetics, New Jersey Medical School-UMDNJ, Newark, New Jersey, USA; and
^* PharmaSeq, Inc., Monmouth Junction, New Jersey, USA

²Correspondence: Department of Microbiology & Molecular Genetics, New Jersey Medical School-UMDNJ, 225 Warren St., P.O. Box 1709, Newark, NJ 07101-1709, USA. E-mail: egoldman{at}umdnj.edu

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 ß-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.

Key Words: E. coli protein synthesis • internal initiation of translation • non-ORF expression • UGA stop codons