Translational options for the pir gene of plasmid R6K: multiple forms of the replication initiator protein pi.
The autogenously controlled pir gene of plasmid R6K was believed to encode a single polypeptide that plays multiple roles in the plasmid's biology. We have isolated an opal (op) mutant at the 18th codon of the pir coding frame which does not totally abolish translation of pir mRNA. In extracts of cells containing this mutation two translational products (35 kDa and 30.2 kDa) have been detected. We propose that the 35-kDa polypeptide produced by the pir18 op mutation contains Trp substituted for Arg18 as the result of an opal readthrough. Translation, which results in the 30.2-kDa polypeptide, originates downstream from the UGA stop signal created by the mutation. Moreover, we realize now that the 30.2-kDa polypeptide is also produced in cells containing a wild-type (wt) pir gene. The shorter variant of the pi protein lacks replication initiation and inhibition functions, as well as autorepressor activity in vivo. We also show that an in-frame fusion of seven N-terminal codons of the trpE gene with a pir gene lacking the first two codons produces two polypeptides which replace the 35-kDa pi protein and are of similar molecular weight. Thus, at least three options exist in the translation of the wt pir mRNA. Start codons are most likely at codon positions 1, 6 or 7, and 36 or 38. Each of these five AUG codons is preceded by a consensus ribosome-binding site (RBS).
York
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D
Department of Bacteriology, University of Wisconsin, Madison 53706.
Ivanov
V
V
Gan
J
J
Filutowicz
M
M
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics*; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008074:Lipoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003062:Codon; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C499290:OppA protein, E coli; D011993:Recombinant Fusion Proteins; C046506:oligopeptide-binding protein, bacteria