CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism

Abstract

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 RPo 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 rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo 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 RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.

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“…In mycobacteria cultured under nutrient-rich conditions, CarD associates with RNAP-promoter complexes throughout the genome to enhance RP o stability (14,58,59,65). Using a bulk fluorescence assay to measure the effects of CarD on transcription initiation kinetics, it was shown that CarD associates with RP o with high affinity and slows the rate of DNA closing by preventing bubble collapse and that CarD associates with RP c with lower affinity and increases the rate of DNA opening (13). Importantly, the concentration of CarD in cells is sufficient for both of these activities to be physiologically relevant (13).…”

“…Using a bulk fluorescence assay to measure the effects of CarD on transcription initiation kinetics, it was shown that CarD associates with RP o with high affinity and slows the rate of DNA closing by preventing bubble collapse and that CarD associates with RP c with lower affinity and increases the rate of DNA opening (13). Importantly, the concentration of CarD in cells is sufficient for both of these activities to be physiologically relevant (13). These two activities of CarD change the kinetics of open complex formation such that the M. bovis RNAP more closely mirrors the E. coli RNAP (13,14).…”

“…Importantly, the concentration of CarD in cells is sufficient for both of these activities to be physiologically relevant (13). These two activities of CarD change the kinetics of open complex formation such that the M. bovis RNAP more closely mirrors the E. coli RNAP (13,14). The interactions between CarD and both DNA and RNAP are required for CarD activity (13).…”

“…The majority of studies on the mechanisms of transcription initiation and its regulation have used Escherichia coli as a model system. However, multiple groups have recently shown that Mycobacterium bovis RNAP, which differs from the M. tuberculosis RNAP by only one amino acid (aa), exhibits an inherently unstable RP o complex compared to E. coli RNAP on the same promoter (13,14). In these reports, saturating …”

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“…It was found that the initiation factors act independently and cooperatively to regulate transcription and that only in the presence of both factors does the transcription kinetics in mycobacteria appear to be similar to that observed in E. coli. 45,46 As both factors are known to play important roles in pathogenesis, global rRNA/gene expression levels and resistance to stress and antibiotics, they would serve as good targets for drug design.…”