Identification of promoter elements in mycobacteria: mutational analysis of a highly symmetric dual promoter directing the expression of replication genes of the Mycobacterium plasmid pAL5000.

The 120 bp origin of replication (ori) for the Mycobacterium plasmid pAL5000 has been shown to comprise the binding sites for the replication protein RepB as well as the start site of transcription for the repA and repB genes, encoding the replication proteins RepA and RepB. In this work it is demonstrated that a third gene product, Rap, is involved in replication in addition to the previously described proteins. Mycobacterium smegmatis cells transformed with replicons carrying the rap gene recover markedly faster upon electroporation than those transformed with the minimal replicon, which lacks rap. The rap gene, oppositely orientated to repA/B, was shown to be transcribed from a promoter orientated back-to-back to and overlapping the repA/B promoter. As a consequence of the extensive dyad symmetry in this region the two promoters share several elements, most of which are situated inside the high-affinity RepB-binding motif in the ori. Transcription of rap runs through the low-affinity RepB-binding site, which is part of the ori and necessary for replication. Both promoters were shown to be repressed by RepB. These divergent promoters were studied through site-specific mutagenesis in a xylE reporter gene assay. The analysis furnished evidence supporting the existence of a distal as well as a proximal element in mycobacterial promoters.