A New Paradigm of Transcriptional Regulation by the SufR-Like Iron-Sulfur Transcription Factors

SufR is an iron-sulfur ([4Fe-4S]) cluster-containing transcription factor belonging to an uncharacterized domain family (COG2345). It has been shown to negatively regulate the sulfur utilization factor (SUF) Fe-S biogenesis system in several Gram-negative and Gram-positive bacteria including Cyanobacteria, Mycobacteria and Streptomyces. The structural basis for its DNA recognition and transcriptional regulation by the SufR-like proteins remains enigmatic. In this study, we present the cryo-EM structure of <i>Mycobacterium tuberculosis</i> SufR bound to its promoter, revealing a new domain architecture. Our structural, biochemical and molecular analyses show that SufR possesses an unusual [4Fe-4S] cluster coordination environment in the sensory domain and recognizes its promoter via a dual-module mechanism using both the AT-hook and the helix-turn-helix (HTH) DNA-binding motif in the DNA-binding domain. This DNA recognition strategy differs from a canonical winged HTH transcription factor. Moreover, our bioinformatic analysis and structural modeling suggest that SufR and SufR-like proteins in the COG2345 family represent a large, previously uncharacterized family of transcription factors widely distributed across bacteria and archaea. Together, these findings establish SufR-like proteins as a new model for transcriptional regulation by Fe-S transcription factors in prokaryotes and uncover the underappreciated evolutionary versatility of AT-hooks.