The PrrAB two-component system (TCS) is essential for Mycobacterium tuberculosis viability. Previously, it was demonstrated that PrrA binds DNA in the absence of PrrB-mediated transphosphorylation and that non-cognate serine/threonine-kinases phosphorylate PrrA threonine-6 (T6). Therefore, we investigated the differential binding affinity and regulatory properties of the M. tuberculosis-derived wild-type PrrA, PrrA phosphomimetic (D58E, T6E), and PrrA phosphoablative (D58A, T6A) proteins with the prrA, dosR, and cydA genes. While we hypothesized greater DNA binding affinity and more pronounced regulation by PrrA phosphomimetic variants, recombinant, wild-type PrrA bound DNA with greatest affinity. Collectively, wild-type PrrA recombinant protein displayed the highest binding affinity to the dosR promoter (K 3.46 ± 2.09 nM), followed by the prrA promoter (K 9.00 ± 2.66 nM). To establish PrrA regulatory activity, we constructed M. smegmatis ΔprrAB::prrA strains with each of the PrrA variants and extrachromosomal prrA, dosR, and cydA promoter-mCherry reporter fusions. Our findings showed that PrrA is autoregulatory and induces dosR expression only during in vitro, hypoxic growth. Combined expression of prrAB in M. smegmatis ΔprrAB significantly induced cydA promoter-mCherry expression. Our studies advanced the understanding of PrrA function and PrrAB phosphorylation-mediated regulatory mechanisms and control of mycobacterial dosR and cydA hypoxic and low-oxygen responsive genes.