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Initial events in bacterial transcription initiation.

Ruff EF, Record MT, Artsimovitch I - Biomolecules (2015)

Bottom Line: At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the -10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP.Intrinsic DNA opening-closing appears less regulated.This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, 1101 University Ave. Madison, Madison, WI 53706, USA. eruff@wisc.edu.

ABSTRACT
Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the -10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

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Mechanism of transcription initiation: resolvable kinetic steps. Minimal mechanisms of open complex formation and dissociation, showing by color coding the steps that contribute to the observed rate constants (kobs, kd) for promoters like λPR that form a stable open complex RPO. For the common experimental situation of excess RNAP, the rate constant for open complex formation (kobs) is determined by the front half of the mechanism (boxed in purple), including the equilibrium constant K1 for formation of the ensemble of closed (I1) intermediates, the forward rate constant k2 of the isomerization step that includes DNA opening, and the excess RNAP concentration. Likewise, kd is determined by the late steps of the mechanism (boxed in green), including the rate constant k-2 for DNA closing and the equilibrium constant K3 for stabilization of the initial open complex I2 to form longer-lived I3 and/or RPO complexes [65,66].
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biomolecules-05-01035-f004: Mechanism of transcription initiation: resolvable kinetic steps. Minimal mechanisms of open complex formation and dissociation, showing by color coding the steps that contribute to the observed rate constants (kobs, kd) for promoters like λPR that form a stable open complex RPO. For the common experimental situation of excess RNAP, the rate constant for open complex formation (kobs) is determined by the front half of the mechanism (boxed in purple), including the equilibrium constant K1 for formation of the ensemble of closed (I1) intermediates, the forward rate constant k2 of the isomerization step that includes DNA opening, and the excess RNAP concentration. Likewise, kd is determined by the late steps of the mechanism (boxed in green), including the rate constant k-2 for DNA closing and the equilibrium constant K3 for stabilization of the initial open complex I2 to form longer-lived I3 and/or RPO complexes [65,66].

Mentions: At λPR opening of the entire 13 bp transcription bubble occurs in the rate-determining isomerization step that forms the first open intermediate, I2 (k2 in Figure 4) [23]. As for DNA melting in solution in the absence of RNAP, this step is highly temperature dependent, increasing 1000-fold between 7 °C and 42 °C with an activation energy of 34 kcal [60,66]. However, unlike in solution, the DNA opening step on RNAP is only weakly dependent on the concentrations of salt and solutes, consistent with a scenario in which it occurs in the local environment of the cleft [64]. The process of transcription bubble closing (k-2 in Figure 4), which is rate determining in dissociation, is even less affected by salt and solute concentration [24] and, at λPR and T7A1, does not appear to be strongly affected by DNA sequence or any modification of RNAP tested thus far, including large deletions [18].


Initial events in bacterial transcription initiation.

Ruff EF, Record MT, Artsimovitch I - Biomolecules (2015)

Mechanism of transcription initiation: resolvable kinetic steps. Minimal mechanisms of open complex formation and dissociation, showing by color coding the steps that contribute to the observed rate constants (kobs, kd) for promoters like λPR that form a stable open complex RPO. For the common experimental situation of excess RNAP, the rate constant for open complex formation (kobs) is determined by the front half of the mechanism (boxed in purple), including the equilibrium constant K1 for formation of the ensemble of closed (I1) intermediates, the forward rate constant k2 of the isomerization step that includes DNA opening, and the excess RNAP concentration. Likewise, kd is determined by the late steps of the mechanism (boxed in green), including the rate constant k-2 for DNA closing and the equilibrium constant K3 for stabilization of the initial open complex I2 to form longer-lived I3 and/or RPO complexes [65,66].
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496709&req=5

biomolecules-05-01035-f004: Mechanism of transcription initiation: resolvable kinetic steps. Minimal mechanisms of open complex formation and dissociation, showing by color coding the steps that contribute to the observed rate constants (kobs, kd) for promoters like λPR that form a stable open complex RPO. For the common experimental situation of excess RNAP, the rate constant for open complex formation (kobs) is determined by the front half of the mechanism (boxed in purple), including the equilibrium constant K1 for formation of the ensemble of closed (I1) intermediates, the forward rate constant k2 of the isomerization step that includes DNA opening, and the excess RNAP concentration. Likewise, kd is determined by the late steps of the mechanism (boxed in green), including the rate constant k-2 for DNA closing and the equilibrium constant K3 for stabilization of the initial open complex I2 to form longer-lived I3 and/or RPO complexes [65,66].
Mentions: At λPR opening of the entire 13 bp transcription bubble occurs in the rate-determining isomerization step that forms the first open intermediate, I2 (k2 in Figure 4) [23]. As for DNA melting in solution in the absence of RNAP, this step is highly temperature dependent, increasing 1000-fold between 7 °C and 42 °C with an activation energy of 34 kcal [60,66]. However, unlike in solution, the DNA opening step on RNAP is only weakly dependent on the concentrations of salt and solutes, consistent with a scenario in which it occurs in the local environment of the cleft [64]. The process of transcription bubble closing (k-2 in Figure 4), which is rate determining in dissociation, is even less affected by salt and solute concentration [24] and, at λPR and T7A1, does not appear to be strongly affected by DNA sequence or any modification of RNAP tested thus far, including large deletions [18].

Bottom Line: At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the -10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP.Intrinsic DNA opening-closing appears less regulated.This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, 1101 University Ave. Madison, Madison, WI 53706, USA. eruff@wisc.edu.

ABSTRACT
Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the -10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

Show MeSH
Related in: MedlinePlus