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 λ o_Lp_L 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 λ DNA fragment which includes the N-unresponsive t_J 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 p_L promoter under N⁺ conditions terminate at t_J between the two genes. S1-nuclease analysis showed that these transcripts terminate at several sites in the t_J region. Similar results were obtained whether the host cells were RNaseIII⁺ or RNaseIII^-. As a control, we showed a complete antitermination of the λ 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.