Structure-function dissection of Myxococcus xanthus CarD N-terminal domain, a defining member of the CarD_CdnL_TRCF family of RNA polymerase interacting proteins.

Paul C. Driscoll,Diego Bernal-Bernal,Gema García-Martínez,S. Padmanabhan,Francisco García-Heras,Montserrat Elías-Arnanz,Aránzazu Gallego-García,María Angeles Jiménez

doi:10.1371/journal.pone.0121322

Paul C. Driscoll, Diego Bernal-Bernal + Show 6 more

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https://doi.org/10.1371/journal.pone.0121322

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Abstract

Two prototypes of the large CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-binding proteins, Myxococcus xanthus CarD and CdnL, have distinct functions whose molecular basis remain elusive. CarD, a global regulator linked to the action of several extracytoplasmic function (ECF) σ-factors, binds to the RNAP β subunit (RNAP-β) and to protein CarG via an N-terminal domain, CarDNt, and to DNA via an intrinsically unfolded C-terminal domain resembling eukaryotic high-mobility-group A (HMGA) proteins. CdnL, a CarDNt-like protein that is essential for cell viability, is implicated in σA-dependent rRNA promoter activation and interacts with RNAP-β but not with CarG. While the HMGA-like domain of CarD by itself is inactive, we find that CarDNt has low but observable ability to activate ECF σ-dependent promoters in vivo, indicating that the C-terminal DNA-binding domain is required to maximize activity. Our structure-function dissection of CarDNt reveals an N-terminal, five-stranded β -sheet Tudor-like domain, CarD1–72, whose structure and contacts with RNAP-β mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-β did not annul activity. Our data suggest that the CarDNt C-terminal segment, CarD61–179, may be structurally distinct from its CdnL counterpart, and that it houses at least two distinct and crucial function determinants: (a) CarG-binding, which is specific to CarD; and (b) a basic residue stretch, which is also conserved and functionally required in CdnL. This study highlights the evolution of shared and divergent interactions in similar protein modules that enable the distinct activities of two related members of a functionally important and widespread bacterial protein family.

Highlights

In the Gram-negative soil bacterium Myxococcus xanthus, a 316-residue global regulatory protein, CarD, is involved in the control of light-induced carotenogenesis, starvation-induced development of multicellular fruiting bodies, and other processes [1,2]
We have shown that CarDNt and CdnL cannot be functionally interchanged in vivo, and while CarD can be deleted in M. xanthus without impairing viability, CdnL is essential for cell growth and survival [11]
We found that the CarDNt stretch spanning residues 61 to 179 (CarD61–179), which is not involved in the interaction with RNA polymerase (RNAP)-β, mediates at least two functionally critical activities: interaction with CarG, and an undefined activity provided by a stretch of basic residues that does not participate in the interaction with CarG

Summary

Introduction

In the Gram-negative soil bacterium Myxococcus xanthus, a 316-residue global regulatory protein, CarD, is involved in the control of light-induced carotenogenesis, starvation-induced development of multicellular fruiting bodies, and other processes [1,2]. In the response to light, CarD is required for the activation of PQRS, a promoter that depends on the alternative extracytoplasmic function σ (ECF-σ) factor CarQ [3]. The second class includes standalone proteins with sizes between 160 and 200 residues, similar in sequence to CarDNt but lacking the characteristic HMGA-like domain of CarD [9]. We named these CdnL (for CarD N-terminal like) to distinguish them from CarD, and because both proteins coexist in M. xanthus [11]. The present study reports our findings with CarDNt, the N-terminal domain of CarD, and counterpart of full-length CdnL

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