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 subunit of RNA polymerase and influences gene expression. The disruption of interactions between CarD and the 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 subunit of RNA polymerase, Mycobacterium tuberculosis CarD (MtbCarD) and the Thermus aquaticus RNA polymerase subunit were recombinantly expressed and purified. Secondary-structure analysis using circular-dichroism spectroscopy indicated that MtbCarD contains $60% -helix, $7% -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.

Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis

“…2C and D). Dimerization of MtbCarD is consistent with the previously reported dimerization of CarD from other mycobacteria [35,36]. When MtbRNAP is pre-incubated with the purified CarD, a heparin-resistant promoter open complex is formed (Fig.…”

CarD integrates three functional modules to promote efficient transcription, antibiotic tolerance, and pathogenesis in mycobacteria

“…Understanding of CarD has hitherto been limited to the RNAP binding activity encoded by the N‐terminus of the protein since the role of the C‐terminus of CarD had remained elusive. Recently, crystal structures of both the Thermus thermophilus CarD and the M. tuberculosis CarD have been solved (Gulten and Sacchettini, ; Srivastava et al ., ; Kaur et al ., ) as well as a crystal structure of the C‐terminus of CarD in isolation of the first 82 amino acids (Gangwar et al ., ). The crystal structures of full‐length CarD confirmed the predicted structure of the N‐terminal RID and revealed a compact helical C‐terminal domain.…”