Structural and biophysical studies on two promoter recognition domains of the extra-cytoplasmic function σ factor σ^C from Mycobacterium tuberculosis

Abstract

σ factors are transcriptional regulatory proteins that bind to the RNA polymerase and dictate gene expression. The extracytoplasmic function (ECF) σ factors govern the environment dependent regulation of transcription. ECF σ factors have two domains σ₂ and σ₄ that recognize the -10 and -35 promoter elements. However, unlike the primary σ factor σ^A, the ECF σ factors lack σ₃, a region that helps in the recognition of the extended -10 element and σ_1.1, a domain involved in the autoinhibition of σA in the absence of core RNA polymerase. Mycobacterium tuberculosis σ^C is an ECF σ factor that is essential for the pathogenesis and virulence of M. tuberculosis in the mouse and guinea pig models of infection. However, unlike other ECF σ factors, σC does not appear to have a regulatory anti-σ factor located in the same operon. We also note that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σ^C2 and σ^C4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σ^C2 and σ^C4 domains of σ^C were determined. These promoter recognition domains are structurally similar to the corresponding domains of σ^A despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σ^C2 as well as surface plasmon resonance measurements reveal that the σ^C2 and σ^C4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σ^C2 is involved in this interdomain interaction. Interaction between the promoter recognition domains in M. tuberculosis σ^C are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor.