Extra Cytoplasmic Function (ECF) sigma factor/regulatory protein (anti-sigma factor) pairs govern environment mediated changes in gene expression in bacteria. The release of the ECF sigma factor from an inactive sigma/anti-sigma factor complex is triggered by specific environmental stimuli. The free sigma factor then associates with the RNA polymerase and drives the expression of genes in its target regulon. Multiple ECF sigma/anti-sigma pairs ensure calibrated changes in the expression profile by correlating diverse environmental stimuli with changes in the intracellular levels of different ECF G factors. Specificity in sigma/anti-sigma factor interaction is thus essential for accurate signal transduction. Here we describe experiments to evaluate interactions between different M. tuberculosis sigma and anti-sigma proteins in vitro. The interaction parameters suggest that cross-talk between non-cognate sigma/anti-sigma pairs is likely. The sequence and conformational determinants that govern interaction specificity in a sigma/anti-sigma complex are not immediately evident due to substantial structural conservation. Sequence-structure analysis of all sigma/anti-sigma pairs suggest that conserved residues are not the primary determinants of sigma/anti-sigma interactions-a finding that suggests a potential route to set tolerance limits in interaction specificity. Non-specific sigma/anti-sigma interactions are likely to be biologically significant as it can contribute to heterogeneity in cellular responses in a bacterial population under less stringent requirements. This finding is relevant for synthetic biology approaches to engineer bacteria using sigma/anti-sigma transcription initiation modules for diverse applications in biotechnology. (C) 2019 Elsevier Inc. All rights reserved.