Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP inEscherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit inMycobacterium tuberculosis . Unlike the well-studiedE. coli RNAP, theM. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution ofM. tuberculosis ω with ω subunits fromE. coli orThermus thermophilus cannot restore the assembly ofM. tuberculosis RNAP. Furthermore, by replacing different regions inM. tuberculosis ω with the corresponding regions fromE. coli ω, we found a nonconserved loop region inM. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) inM. tuberculosis RNAP but not inE. coli orT. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD inM. tuberculosis RNAP destabilized the binding ofM. tuberculosis ω on RNAP and compromisedM. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly inM. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role ofM. tuberculosis ω and highlighted the importance of the ω loop region inM. tuberculosis RNAP assembly.IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. InEscherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogenMycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.