ABSTRACT
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 in
Escherichia coli
and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in
Mycobacterium tuberculosis
. Unlike the well-studied
E. coli
RNAP, the
M. tuberculosis
RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of
M. tuberculosis
ω with ω subunits from
E. coli
or
Thermus thermophilus
cannot restore the assembly of
M. tuberculosis
RNAP. Furthermore, by replacing different regions in
M. tuberculosis
ω with the corresponding regions from
E. coli
ω, we found a nonconserved loop region in
M. 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) in
M. tuberculosis
RNAP but not in
E. coli
or
T. thermophilus
RNAP is close to the ω loop region. Deletion of this β′CTD in
M. tuberculosis
RNAP destabilized the binding of
M. tuberculosis
ω on RNAP and compromised
M. tuberculosis
core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in
M. 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 of
M. tuberculosis
ω and highlighted the importance of the ω loop region in
M. tuberculosis
RNAP assembly.
IMPORTANCE
DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α
2
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. In
Escherichia 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 pathogen
Mycobacterium 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.
Mass Spectrometry of Escherichia coli RNA Polymerase: Interactions of the Core Enzyme with σ70 and Rsd Protein