DksA2, a zinc-independent structural analog of the transcription factor DksA - PubMed (original) (raw)
DksA2, a zinc-independent structural analog of the transcription factor DksA
Ran Furman et al. FEBS Lett. 2013.
Abstract
Transcription factor DksA contains a four-Cys Zn(2 +)-finger motif thought to be responsible for structural integrity and the relative disposition of its domains. Pseudomonas aeruginosa encodes an additional DksA paralog (DksA2) that is expressed selectively under Zn(2+) limitation. Although DksA2 does not bind Zn(2+), it complements the Escherichia coli dksA deletion and has similar effects on transcription in vitro. In this study, structural and biochemical analyses reveal that DksA2 has a similar fold, domain structure and RNA polymerase binding properties to those of the E. coli DksA despite the lack of the stabilizing metal ion.
Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Figures
Fig. 1
Structures of (A) PA DksA2 and (B) EC DksA (1TJL) with the key residues shown as sticks. The Zn2+ ion is shown as a sphere, the C-termini are marked.
Fig. 2
DksA2 and DksA bind to RNAP similarly. (A) Binding through the secondary channel was assayed using localized Fe2+ cleavage as described in [16] using 20 nM [32P]-labeled DksA or DksA2 and 400 nM E. coli RNAP (WT or the RHDD variant). (B) Effects of mutations in DksA2 and RNAP on transcription from the rrnB P1 promoter. Assays were performed in three repeats as described in [6] with 5 µMDksA2, 30 nM RNAP, and 10 nM template; ApC, UTP and [α32P]-GTP were used as substrates. Gels were quantified by phosphorimaging (ImageQuant software, Molecular Dynamics).
Fig. 3
A disulfide bond in DksA2. (A) A bridge between Cys96 and Cys117 observed in the structure. (B) Probing the thiols with [PEG]12-maleimide. Labeling was done for 2 h at room temperature and samples were analyzed on a 12% SDS–PAGE gel. (C) DksA2 activity is modestly increased in buffers containing BME. Transcription from the rrnB P1 promoter was assayed as described in Fig. 2; the fold effect corresponds to a fraction of overall transcription in the absence of DksA. (D) Oxidation has a modest inhibitory effect on DksA2 activity. (E) DksA2 cysteines are mostly in a reduced form in the cell. Cell were treated with IAM prior to lysis. DksA2 was purified and treated with [PEG]12, as above.
Fig. 4
Effects of substitutions of DksA2 cysteines. (A) Substitutions of Cys96 and Cys117 abolish DksA2 activity at rrnB P1. (B) Substitutions of two Cys residues have minimal effect on DksA2 secondary structure as monitored by circular dichroism, featuring strong helical signatures at 208 and 222 nm. (C) Intrinsic protein fluorescence emission of DksA2WT and DksA2C96V,117V (200 nM) in 20 mM HEPES, pH 7.5, 50 mM NaCl. Excitation was done at 285 nm and emission scan was done between 320 and 380 nm with a 5 nm slit size using F7000 Fluorimeter (Hitachi). (D) Overlay of two-dimensional 15N–1H HSQC spectra of DksA2 (black) and DksA2C96V (red). Loss of dispersed resonances in the DksAC96V spectrum suggests destabilization of the globular domain.
References
- Blencowe DK, Morby AP. Zn(II) metabolism in prokaryotes. FEMS Microbiol. Rev. 2003;27:291–311. -PubMed
- Perederina A, Svetlov V, Vassylyeva MN, Tahirov TH, Yokoyama S, Artsimovitch I, Vassylyev DG. Regulation through the secondary channel-structural framework for ppGpp-DksA synergism during transcription. Cell. 2004;118:297–309. -PubMed
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