Publication Date:
2015-04-02
Description:
CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RP o formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnA P3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RP o that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo . In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RP o on rrnA P3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.
Print ISSN:
0305-1048
Electronic ISSN:
1362-4962
Topics:
Biology
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