An intrinsically disordered domain of transcription terminator Rho governs genome-wide Rho utilization (Rut) site selection in Mycobacterium tuberculosis.

Rho-dependent transcription termination (RDTT) is crucial for bacterial gene regulation, fitness, and pathogenicity. RDTT relies on the ATP-dependent Rho helicase, which engages nascent transcripts at loosely defined, C > G-biased Rho utilization (Rut) sites to dismantle transcription complexes. In Mycobacterium tuberculosis, Rho features a unique, large N-terminal insertion domain (NID) of uncertain function. Using enzymatic Helicase-SELEX screening, we identified 1759 putative Rut sites across the M. tuberculosis genome with wild-type Rho (MtbRho) but only 47 with a NID-deleted variant (ΔNID-MtbRho), demonstrating NID's importance for productive substrate recruitment. The selected Rut sites display an unusual composition and frequently occur upstream of or within coding regions, implying that MtbRho plays a broad role in conditional gene regulation. Integration with transcriptomics-defined RNA 3' ends further highlights the contribution of MtbRho to transcriptional plasticity, through both direct termination and downstream regulatory cascades. Complementary RDTT assays, affinity measurements, solution NMR, and cryogenic electron microscopy analyses show that the NID is intrinsically disordered, yet critical for engaging Rut sites with substantial secondary structure. Altogether, our findings provide the first genome-wide map and characterization of Rut sites in a GC-rich pathogen and define the NID as a model intrinsically disordered protein region with a critical mechanistic and functional role in mycobacterial gene expression.