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Structure of the Mtb CarD/RNAP ?-lobes complex reveals the molecular basis of interaction and presents a distinct DNA-binding domain for Mtb CarD.

ABSTRACT: CarD from Mycobacterium tuberculosis (Mtb) is an essential protein shown to be involved in stringent response through downregulation of rRNA and ribosomal protein genes. CarD interacts with the ?-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 ?-subunit ?1 and ?2 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 ?1 structure. Interestingly, the structure of the C-terminal domain, which is required for complete CarD function in vivo, represents a distinct DNA-binding fold.

SUBMITTER: Gulten G

PROVIDER: S-EPMC3894638 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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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.

Gulten Gulcin G Sacchettini James C JC

Structure (London, England : 1993) 20130919 10

CarD from Mycobacterium tuberculosis (Mtb) is an essential protein shown 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 ...[more]

PMID: 24055315

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Project description:Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP ? subunit (?CTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in ?CTD severely impaired Spx-activated transcription. Attempts to substitute alanine for ?CTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where "Y263C" indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound ?CTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli ?D259, which has been implicated in ?CTD-?70 interaction (?70 R603, corresponding to R362 of B. subtilis ?A). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while ?CTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-?CTD complex.IMPORTANCE Though extensively studied in Escherichia coli, the role of ?CTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis Here, we conduct phenotypic analyses of putatively lethal ?CTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-?CTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several ?CTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

Dataset Information

Structure of the Mtb CarD/RNAP ?-lobes complex reveals the molecular basis of interaction and presents a distinct DNA-binding domain for Mtb CarD.

Publications

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.

Similar Datasets

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