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Key roles of the downstream mobile jaw of Escherichia coli RNA polymerase in transcription initiation.

ABSTRACT: Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free energy to unwind and separate 13 base pairs of ?P(R) promoter DNA to form the unstable open intermediate I(2), which rapidly converts to much more stable open complexes (I(3), RP(o)). Conversion of I(2) to RP(o) involves folding/assembly of several mobile RNAP domains on downstream duplex DNA. Here, we investigate effects of a 42-residue deletion in the mobile ?' jaw (?JAW) and truncation of promoter DNA beyond +12 (DT+12) on the steps of initiation. We find that in stable ?JAW open complexes the downstream boundary of hydroxyl radical protection shortens by 5-10 base pairs, as compared to wild-type (WT) complexes. Dissociation kinetics of open complexes formed with ?JAW RNAP and/or DT+12 DNA resemble those deduced for the structurally uncharacterized intermediate I(3). Overall rate constants (k(a)) for promoter binding and DNA opening by ?JAW RNAP are much smaller than for WT RNAP. Values of k(a) for WT RNAP with DT+12 and full-length ?P(R) are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RP(o) formation, while downstream DNA functions primarily as the assembly platform after DNA opening.

SUBMITTER: Drennan A

PROVIDER: S-EPMC3517728 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Key roles of the downstream mobile jaw of Escherichia coli RNA polymerase in transcription initiation.

Drennan Amanda A Kraemer Mark M Capp Michael M Gries Theodore T Ruff Emily E Sheppard Carol C Wigneshweraraj Sivaramesh S Artsimovitch Irina I Record M Thomas MT

Biochemistry 20121114 47

Differences in kinetics of transcription initiation by RNA polymerase (RNAP) at different promoters tailor the pattern of gene expression to cellular needs. After initial binding, large conformational changes occur in promoter DNA and RNAP to form initiation-capable complexes. To understand the mechanism and regulation of transcription initiation, the nature and sequence of these conformational changes must be determined. Escherichia coli RNAP uses binding free energy to unwind and separate 13 b ...[more]

PMID: 23116321

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Project description:Transcription by all RNA polymerases (RNAPs) requires a series of large-scale conformational changes to form the transcriptionally competent open complex RP(o). At the lambdaP(R) promoter, Escherichia coli sigma(70) RNAP first forms a wrapped, closed 100 bp complex I(1). The subsequent step opens the entire DNA bubble, creating the relatively unstable (open) complex I(2). Additional conformational changes convert I(2) to the stable RP(o). Here we probe these events by dissecting the effects of Na(+) salts of Glu(-), F(-), and Cl(-) on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I(1) at 25 degrees C is approximately 30-fold larger in Glu(-) than in Cl(-) at the same Na(+) concentration, with the same log-log salt concentration dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I(1) to I(2)) are only weakly dependent on salt concentration, and the opening rate constant is insensitive to replacement of Cl(-) with Glu(-). These very small effects of salt concentration on a process (DNA opening) that is strongly dependent on salt concentration in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process and/or that compensation is present between ion uptake and release. Replacement of Cl(-) with Glu(-) or F(-) at 25 degrees C greatly increases the lifetime of RP(o) and greatly reduces its salt concentration dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu(-) and F(-) drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I(2) to RP(o), while Cl(-) does not. Because the Hofmeister effect of Glu(-) or F(-) largely compensates for the destabilizing Coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RP(o) remains long-lived even at 0.5 M Na(+) in Glu(-) or F(-) salts. The observation that Esigma(70) RP(o) complexes are exceedingly long-lived at moderate to high Glu(-) concentrations argues that Esigma(70) RNAP does not dissociate from strong promoters in vivo when the cytoplasmic glutamate concentration increases during osmotic stress.

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Key roles of the downstream mobile jaw of Escherichia coli RNA polymerase in transcription initiation.

Publications

Key roles of the downstream mobile jaw of Escherichia coli RNA polymerase in transcription initiation.

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