Regulation of the gyr operon of Mycobacterium tuberculosis by overlapping promoters, DNA topology, and reiterative transcription

Abstract

DNA gyrase introduces negative supercoils into DNA to maintain topological homeostasis. The genes encoding gyrase, gyrB and gyrA, form a dicistronic operon in Mycobacterium tuberculosis (Mtb) and other actinobacteria. Earlier work indicated that DNA relaxation stimulates the expression of the gyr genes, a phenomenon termed relaxation-stimulated transcription (RST). The present study addresses the underlying mechanism of gyr operon regulation. The operon is regulated by overlapping and divergently oriented promoters located upstream of gyrB. The principal promoter, P_gyrB1, drives transcription of the operon, while a weak "reverse" promoter, P_gyrR, transcribes in opposite direction. We demonstrate that P_gyrR plays a role in fine tuning gyr gene expression by reiterative transcription (RT), a regulatory mechanism hitherto not found in Mtb. In vitro transcription assays showed that RT at P_gyrR depended on the negatively supercoiled state of the DNA template. The principal promoter, P_gyrB1, was also sensitive to DNA supercoiling, but it was stimulated by DNA relaxation. Moreover, RNA polymerase binding to the promoter was efficient at P_gyrB1 when template DNA was relaxed, whereas binding to P_gyrR was preferred when DNA was supercoiled. Thus, a collaboration between RST and RT governs the regulation of the gyr operon; the differing sensitivity of the two overlapping promoters to superhelix density explains how gyrase expression responds to changes in supercoiling to determine the efficiency of transcription initiation.

Keywords: DNA gyrase; Mycobacterium; Overlapping promoters; Reiterative transcription; Relaxation stimulated transcription; Topology.