Expression of the repA1 gene of IncFII plasmid NR1 is translationally coupled to expression of an overlapping leader peptide.
Examination of a group of mutants of plasmid NR1 that had lost the expression of IncFII plasmid incompatibility (Inc-) revealed a group that had also lost replication proficiency (Rep-). These mutants were obtained from plasmids in which the NR1 replication control region was present in a cointegrate with plasmid pBR322. Whereas the wild-type parental cointegrate plasmid was capable of replicating in a polA host owing to the PolA independence of NR1 replication, the mutants were not able to transform a polA host. Losses of both expression of IncFII plasmid incompatibility and replication proficiency were found to result from the same single base-pair substitution in four independently isolated Inc- Rep- mutants. The mutation inactivates promoter PE for the transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. Although the loss of RNA-E synthesis had been expected to increase the expression of repA1, the efficiency of translation of repA1 mRNA from these mutants was at least 100-fold lower than that from the wild type, as revealed by repA1-lacZ translational fusions. The PE mutation introduced a stop codon into a 24-amino-acid reading frame that precedes the repA1 gene and terminates just 2 bp downstream from the repA1 start codon. This putative leader peptide was also expressed in a lacZ translational fusion, and its expression was reduced by a factor of 10(4) by the PE mutation. The expression of the leader peptide and the expression of repA1 were regulated by RNA-E. These results suggest that the expression of repA1 is coupled to the translation of the leader peptide and that the repression of repA1 translation by RNA-E may occur via inhibition of the translation of the leader peptide.
Wu
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Center for Molecular Biology, Wayne State University, Detroit, Michigan 48202.
Wang
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Womble
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Rownd
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; C070013:REPA1 protein, Bacteria