title: Structure of the carboxy-terminal domain of Mycobacterium tuberculosis CarD protein: an essential rRNA transcriptional regulator.
abstract: 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 (CarD(83-161)) was crystallized and its X-ray structure was determined at 2.1 Å resolution. CarD(83-161) 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 CarD(83-161) 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 CarD(83-161) structure. The structural changes in the CarD(83-161) structure occurred owing to proteolysis and crystallization artifacts.
authors.affiliation: Structural Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
authors.forename: Shanti P
authors.initials: SP
authors.lastname: Gangwar
authors.affiliation: Structural Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
authors.forename: Sita R
authors.initials: SR
authors.lastname: Meena
authors.affiliation: Structural Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
authors.forename: Ajay K
authors.initials: AK
authors.lastname: Saxena
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012335:RNA, Ribosomal / Q000235:genetics*; D013056:Spectrophotometry, Ultraviolet; D016963:Thermus thermophilus / Q000737:chemistry; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D012335:RNA, Ribosomal
keywords: CarD binding analysis; Mycobacterium tuberculosis CarD; Thermus aquaticus RNAP β subunit