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The CarD protein is highly expressed in mycobacterial strains under basal conditions and is transcriptionally induced during multiple types of genotoxic stress and starvation. The CarD protein binds the β subunit of RNA polymerase and influences gene expression. The disruption of interactions between CarD and the β subunit of RNA polymerase has a significant effect on mycobacterial survival, resistance to stress and pathogenesis. To understand the structure of CarD and its interaction with the β subunit of RNA polymerase, Mycobacterium tuberculosis CarD (MtbCarD) and the Thermus aquaticus RNA polymerase β subunit were recombinantly expressed and purified. Secondary-structure analysis using circular-dichroism spectroscopy indicated that MtbCarD contains ∼60% α-­helix, ∼7% β-sheet and ∼33% random-coil structure. The C-terminal domain of MtbCarD (CarD83–161) was crystallized and its X-ray structure was determined at 2.1 Å resolution. CarD83–161 forms a distorted Y-shaped structure containing bundles of three helices connected by a loop. The residues forming the distorted Y-shaped structure are highly conserved in CarD sequences from other mycobacterial species. Comparison of the CarD83–161 structure with the recently determined full-length M. tuberculosis and T. thermophilus CarD crystal structures revealed structural differences in residues 141–161 of the C-­terminal domain of the CarD83–161 structure. The structural changes in the CarD83–161 structure occurred owing to proteolysis and crystallization artifacts.

Supporting information

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Tagged Image Format File (TIF) image https://doi.org/10.1107/S2053230X13034407/gx5219sup1.tif
Supplementary Fig. S1. Circular-dichroism analysis of full-length MtbCarD. (A) CD spectra of MtbCarD were recorded from 260 to 200 nm at 250C and normalized to the mean residue ellipticity. The secondary-structure content of MtbCarD was quite similar to the secondary-structure content obtained from theoretical prediction analysis. (B) PSIPRED analysis of full-length MtbCarD.

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Portable Document Format (PDF) file https://doi.org/10.1107/S2053230X13034407/gx5219sup2.pdf
Supplementary Table S1. N-terminal sequencing of CarD protein obtained from dissolved crystals used for the X-ray diffraction experiment.

PDB reference: CarD83–161, 4kmc


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