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

Rammohan J, Ruiz Manzano A, Garner AL, Stallings CL, Galburt EA - Nucleic Acids Res. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.

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RNAP concentration dependence of promoter opening. (A) Fluorescence enhancement as a function of time for increasing concentrations of EcoRNAP (0–238 nM). (B) Fluorescence enhancement as a function of time for increasing concentrations of MboRNAP (0–600 nM). (C) Equilibrium fluorescence fold change for both EcoRNAP and MboRNAP as a function of polymerase concentration. Fits (solid lines) of the data allow the extraction of concentrations of half-maximal effect (23 ± 5 nM and 212 ± 43 nM) and saturated fluorescence enhancements (1.44 and 0.28) for EcoRNAP and MboRNAP, respectively.
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Figure 2: RNAP concentration dependence of promoter opening. (A) Fluorescence enhancement as a function of time for increasing concentrations of EcoRNAP (0–238 nM). (B) Fluorescence enhancement as a function of time for increasing concentrations of MboRNAP (0–600 nM). (C) Equilibrium fluorescence fold change for both EcoRNAP and MboRNAP as a function of polymerase concentration. Fits (solid lines) of the data allow the extraction of concentrations of half-maximal effect (23 ± 5 nM and 212 ± 43 nM) and saturated fluorescence enhancements (1.44 and 0.28) for EcoRNAP and MboRNAP, respectively.

Mentions: We first studied the concentration dependence of EcoRNAP (σ70) and MboRNAP (σA) holoenzymes on open-complex formation on the M. tuberculosis rrnAP3 promoter at 25°C. Fluorescence enhancements after the addition of 2–283 nM EcoRNAP (Figure 2A) or 37.5–600 nM MboRNAP (Figure 2B) were monitored for 20 min. Final fold enhancements were plotted versus RNAP holoenzyme concentration and fit to extract a concentration at which the enhancement is half-maximal (Keff, Figure 2C). As expected, the amount of open complex increases as concentration increases for both forms of RNAP, however EcoRNAP exhibits a Keff almost 10-fold smaller than MboRNAP (23 ± 5 nM versus 212 ± 43 nM, 95% confidence bounds). More strikingly, at saturating concentrations of the respective polymerases, the fluorescence enhancement for EcoRNAP is 1.44 ± 0.1 while MboRNAP only reaches an enhancement of 0.30 ± 0.1. The enhancement for EcoRNAP is comparable to that described previously for EcoRNAP on a consensus promoter (∼1.4) (17), suggesting that this signal is indicative of fully open DNA (i.e. 100% open complex) on rrnAP3. By comparison, MboRNAP σA holoenzyme, even when fully occupying the DNA template at saturating concentrations of polymerase, is not capable of opening a large percentage of the promoters leaving the majority of bound complexes in the closed state. This demonstrates that MboRNAP forms a significantly less stable open complex as compared to EcoRNAP, even on its own mycobacterial promoter.


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

Rammohan J, Ruiz Manzano A, Garner AL, Stallings CL, Galburt EA - Nucleic Acids Res. (2015)

RNAP concentration dependence of promoter opening. (A) Fluorescence enhancement as a function of time for increasing concentrations of EcoRNAP (0–238 nM). (B) Fluorescence enhancement as a function of time for increasing concentrations of MboRNAP (0–600 nM). (C) Equilibrium fluorescence fold change for both EcoRNAP and MboRNAP as a function of polymerase concentration. Fits (solid lines) of the data allow the extraction of concentrations of half-maximal effect (23 ± 5 nM and 212 ± 43 nM) and saturated fluorescence enhancements (1.44 and 0.28) for EcoRNAP and MboRNAP, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: RNAP concentration dependence of promoter opening. (A) Fluorescence enhancement as a function of time for increasing concentrations of EcoRNAP (0–238 nM). (B) Fluorescence enhancement as a function of time for increasing concentrations of MboRNAP (0–600 nM). (C) Equilibrium fluorescence fold change for both EcoRNAP and MboRNAP as a function of polymerase concentration. Fits (solid lines) of the data allow the extraction of concentrations of half-maximal effect (23 ± 5 nM and 212 ± 43 nM) and saturated fluorescence enhancements (1.44 and 0.28) for EcoRNAP and MboRNAP, respectively.
Mentions: We first studied the concentration dependence of EcoRNAP (σ70) and MboRNAP (σA) holoenzymes on open-complex formation on the M. tuberculosis rrnAP3 promoter at 25°C. Fluorescence enhancements after the addition of 2–283 nM EcoRNAP (Figure 2A) or 37.5–600 nM MboRNAP (Figure 2B) were monitored for 20 min. Final fold enhancements were plotted versus RNAP holoenzyme concentration and fit to extract a concentration at which the enhancement is half-maximal (Keff, Figure 2C). As expected, the amount of open complex increases as concentration increases for both forms of RNAP, however EcoRNAP exhibits a Keff almost 10-fold smaller than MboRNAP (23 ± 5 nM versus 212 ± 43 nM, 95% confidence bounds). More strikingly, at saturating concentrations of the respective polymerases, the fluorescence enhancement for EcoRNAP is 1.44 ± 0.1 while MboRNAP only reaches an enhancement of 0.30 ± 0.1. The enhancement for EcoRNAP is comparable to that described previously for EcoRNAP on a consensus promoter (∼1.4) (17), suggesting that this signal is indicative of fully open DNA (i.e. 100% open complex) on rrnAP3. By comparison, MboRNAP σA holoenzyme, even when fully occupying the DNA template at saturating concentrations of polymerase, is not capable of opening a large percentage of the promoters leaving the majority of bound complexes in the closed state. This demonstrates that MboRNAP forms a significantly less stable open complex as compared to EcoRNAP, even on its own mycobacterial promoter.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.

Show MeSH
Related in: MedlinePlus