Plasmid vectors designed for the analysis of transcription termination signals.

We have constructed synthetic operons in which two genes (cat and lacZ or cat and galK) were placed in tandem under the control of the bacteriophage lambda oLpL operator and promoter. Restriction sites were introduced between the promoter and the proximal cat gene or between the cat and lacZ or galK genes. In the latter case, introduction of a transcriptional terminator between the two structural genes should affect only the distal gene. Thus, following induction, the expression of the cat gene serves as an internal control, compensating for changes due to plasmid copy number or possible decrease in transcription initiation. We used these plasmids to select a lambda DNA fragment which includes the N-unresponsive tJ transcriptional terminator. This DNA fragment was inserted between the cat and galK genes. Enzymatic assays of these two gene activities following induction indicate that transcripts initiated at the pL promoter under N+ conditions terminate at tJ between the two genes. S1-nuclease analysis showed that these transcripts terminate at several sites in the tJ region. Similar results were obtained whether the host cells were RNaseIII+ or RNaseIII-. As a control, we showed a complete antitermination of the lambda t'I terminator under similar conditions, indicating that a sufficient amount of the N gene product is made from one N gene copy to suppress terminators carried on multicopy plasmids.