Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.
An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.
Goldman
E
E
Department of Microbiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA. egoldman@umdnj.edu
Korus
M
M
Mandecki
W
W
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D018388:Codon, Terminator; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli; D016368:Frameshift Mutation; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D014176:Protein Biosynthesis; D016364:Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D036022:Capsid Proteins; D002352:Carrier Proteins; D018388:Codon, Terminator; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D001616:beta-Galactosidase; C051815:somatotropin-binding protein