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Relatively few rho-dependent terminators have been described in the literature. This manuscript describes another such terminator, isolated from phage T5. Functional analysis, involving the generation of deletion subclones, has permitted the localization of the terminator on a 413 bp fragment. Attempts to further reduce the size of this fragment resulted in loss of terminator activity. DNA sequence analysis of the terminator region supports the model whereby a rho-dependent terminator is composed of a long region of non-translated unstructured DNA, which permits rho binding, followed by RNA polymerase pausing sites where termination (in the presence of rho) may occur. The results agree with the currently held hypothesis that, despite the many similarities found between various rho dependent termination sequences, no consensus can be defined for either the rho binding or the rho termination sites (1,2).

Cloning into the HindIII site of plasmid pBR322 inactivates the tetR promoter and usually prevents the expression of the tetR gene. The corresponding clones revert to tetracycline resistance at a low frequency. Such reversion is caused by gene rearrangement within the plasmids. DNA sequence analysis reveals three classes of revertants. The first class contains plasmids with partial duplications, which result in the fusion of the promoter of the RNA I species to the tetR gene. The event itself destroys the region encoding the RNA primer for replication and thus the plasmids would be replication defective if the duplication did not also include this region of the molecule. The plasmids from the second class are simple deletions which again fuse the tetR region to the RNA I promoter. In one case, the junction takes place at the end of the RNA I transcript, leaving RNA I and the RNA primer virtually intact. However, it removes the promoter of the RNA primer, the latter now being read from the cloned material. The second member of this class has fused the tetR gene well upstream of the RNA I region so that the RNA primer is still read from its own promoter. The low-level tetracycline resistance is probably due to partial read-through of the RNA I terminator. The third class of revertants differs from the previous two by the acquisition of foreign DNA in the form of an IS2-type insertion element which is known to promote transcription.

Transcription promoters and terminators have been cloned from the late region of bacteriophage T5 DNA and their strengths determined in vivo in plasmid derivatives. DNA sequence analysis shows these transcription signals to be remarkable in that, in all four cases studied in detail, the promoters and terminators overlapped or were very close together.