Analysis of ColE1 expression in vitro after chromosome fragmentation

Abstract

The RNA and protein products synthesized from ColE1 DNA were observed before and after cutting the DNA with different restriction enzymes. Synthesis was carried out in the DNA-directed coupled transcription translation system. The S-30 extracts used to catalyze synthesis were prepared from a recB mutant in which the linear DNA fragments resulting from restriction enzyme cleavage were spared from the usual degradation by exonucleolytic attack. By correlating the observed in vitro synthesized products with the location of the cleavage sites in the plasmid chromosome, it was possible to identify specific gene products. The col gene catalyzes the synthesis of numerous peptides in addition to the 56-kilodalton colicin protein encoded by this gene. Most of the subsidiary products appear to arise as the result of premature termination by a mechanism(s) which remains to be determined. A unique RNA and protein were characterized as products of the imm gene. The RNA has an estimated mass of 150 kilodaltons, and the protein has an estimated mass of 13 kilodaltons. From the DNA sequence of the chromosome, it was concluded that the transcripts from the imm and col genes must crisscross each other over a region of about 75 base pairs. Such a pattern of transcription might lead to interference of transcription of one gene by the other gene. Consistent with this hypothesis, it was found that imm gene transcription increased severalfold in vitro when the chromosome was cleaved in a way that eliminated transcription originating at the col gene promoter. Surprisingly, the increase in transcription by this mechanism did not result in a significant increase in the synthesis of the imm gene-encoded protein.