Analysis of the terminator region after thedeoCABDopcron ofEscherichia coliK-12 using a new class of single copy number operon-fusion vectors

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Abstract
We describe the construction of low copy number operon-fusion vectors, and use one of these vectors for the cloning and transcriptional analysis of the terminator region after the deo operon of Escherichia coli K-12. The new vectors are miniderivatives of plasmid Rl containing the parB stability locus of this plasmid and the lac genes as a selectable marker. Since the copy number of the vectors is only one per genome-equivalent at temperatures below 37°C this system is ideally suited for isolation and characterization of transcriptional and translational signals from E. coli. Our results show that a very strong terminator (deot), which resembles Rho-independent terminators, is located 60 bp downstream from the fourth structural gene of the deo operon. This confirms that deoD is the last gene in the operon. In addition, we have identified a new promoter just after the deot terminator and a short DNA sequence that is able to reduce lacZ expression by 85% when inserted between the deoP2 promoter and the lac genes.