Mycobacteriophage D29 is a bacteriophage that infects mycobacteria. In the genome of this phage, there exist multiple copies of a regulatory sequence known as ‘stoperator’ that bind the phage repressor. The ultimate consequence of repressor-stoperator interaction is that phage gene expression comes to a halt and progression of the lytic pathway is inhibited. While investigating the transcriptome of the mycobacteriophage D29, we found that several of these stoperators also functioned as promoters.

The basis for the dual nature of the stoperators was investigated by performing reporter assays, in vitro transcription and DNA binding assays, and finally mutational analysis. The results indicate that the perceived duality of a subset of the stoperators is due to the presence of a conserved motif (5’-TTGACA-3’) that is commonly known as -35 conserved element in the context of eubacterial promoters. Presence of this motif enables stoperators to interact with the phage repressor as well as with sigma factors, in a mutually exclusive manner. Thus two transcriptional factors that function in a diametrically opposite manner recognize a common sequence motif. Further, both repressors and sigma factors have a helix-turn-helix motif in common. Based on the findings presented in this study, we speculate how promoter elements may have evolved from simpler regulatory modules similar to stoperators. We propose that in the ancient past, simple DNA-protein interaction modules may have existed in which the sequence motif 5'-TTGACA-3' played a key. This motif formed the recognition site of helix-turn-helix family of regulators. In the later stages of evolution, both the recognition site and/or their protein interactors may have evolved further resulting in diversification. The sigma factor region 4 and its binding site are likely to have originated from one such module. In our view, the repressor Gp71 represents an ancestral version of region 4 of sigma factors.