Structure of the Mtb CarD/RNAP β-Lobes Complex Reveals the Molecular Basis of Interaction and Presents a Distinct DNA-Binding Domain for Mtb CarD

Abstract

SUMMARY CarD from Mycobacterium tuberculosis (Mtb) is an essential protein thought to be involved in stringent response through downregulation of rRNA and ribosomal protein genes. CarD interacts with the β-subunit of RNAP and this interaction is vital for Mtb’s survival during the persistent infection state. We have determined the crystal structure of CarD in complex with the RNAP β-subunit β1 and β2 domains at 2.1 Å resolution. The structure reveals the molecular basis of CarD/RNAP interaction, providing a basis to further our understanding of RNAP regulation by CarD. The structural fold of the CarD N-terminal domain is conserved in RNAP interacting proteins such as TRCF-RID and CdnL, and displays similar interactions to the predicted homology model based on the TRCF/RNAP β1 structure. Interestingly, the structure of the C-terminal domain, which is required for complete CarD function in vivo, represents a novel DNA binding fold.

Mycobacterium tuberculosis Transcription Machinery: Ready To Respond to Host Attacks

“…This broad panel of factors allows M. tuberculosis to tune its transcriptional response for a large and diverse set of conditions. All of the factors in M. tuberculosis belong to the 70 family, whose members in E. coli recognize two sequences in the promoter DNA, the Ϫ10 element (recognized by sigma region 2.4) and the Ϫ35 element (recognized by sigma region 4.2) (19). M. tuberculosis promoters contain a conserved Ϫ10 sequence that is essential and sometimes sufficient for transcription, while the Ϫ35 sequences are less conserved (19)(20)(21).…”

“…The housekeeping factor A has been reported to have an affinity for M. tuberculosis RNAP core enzyme similar to that of the alternative factor F (74), in which case RbpA may be necessary to improve A affinity and competitiveness for RNAP under conditions that require the activity of A . In E. coli, in contrast, 70 has a very high affinity to the RNAP core enzyme and thus can outcompete other factors under conditions where it is required without accessary factors such as RbpA. The RbpA SID and BL are important and sufficient to partially activate transcription in vitro (71,72), but full activation of transcription requires the full-length protein, although the function of the N terminus of RbpA remains elusive.…”

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.…”

Bacterial RNA Polymerase-DNA Interaction—The Driving Force of Gene Expression and the Target for Drug Action

“…CarD is widely distributed in at least ten bacterial phyla, including Firmicutes, Cyanobacteria, Actinobacteria, and Deinococcus-Thermus (Bae et al, 2015a), but is absent in γ-Proteobacteria such as Eco . The structure and function of Mtb and Tth CarD have been characterized (Stallings et al, 2009; Gulten and Sacchettini, 2013; Srivastava et al, 2013), and the molecular mechanism of its action was recently proposed based on the 3-D structure of Tth CarD in complex with RPo assembled on consensus promoter DNA with full transcription bubble (Bae et al, 2015a). …”