Structure-Function Dissection of Myxococcus xanthus CarD N-Terminal Domain, a Defining Member of the CarD_CdnL_TRCF Family of RNA Polymerase Interacting Proteins

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.

Identification of CdnL, a Putative Transcriptional Regulator Involved in Repair and Outgrowth of Heat-Damaged Bacillus cereus Spores

“…CdnL (BC4714) belongs to CarD_CdnL_TRCF family of bacterial RNA polymerase-binding proteins. A family archetype, CarD, is associated with carotenogenesis and fruiting body formation in Myxococcus xanthus , and is one of two prokaryotic examples of high-mobility group A (HMGA) proteins [ 47 – 49 ]. The C-terminus of CarD contains a HMGA domain responsible for DNA binding and the N-terminus contains a transcription repair coupling factor (TRCF) domain that shares a binding site with the RNAP β subunit [ 50 ].…”

“…A more common member of the bacterial CarD family is represented by the CarD N-terminal like protein (CdnL) that lacks a DNA-binding domain and is also present in M . xanthus and Mycobacterium tuberculosis where it has an important role in stress resistance [ 49 ]. In particular, the M .…”

Understanding molecular consequences of putative drug resistant mutations in Mycobacterium tuberculosis

“…SNP data and association p -values were available from a GWAS of 6,465 clinical isolates, which had drug susceptibility test data for rifampicin, isoniazid and D-cycloserine 24 . Simultaneously we compiled a list of all the anti-TB drugs and their respective targets from the literature, and a search for suitable crystallographic structures was initially made in PDBe 48 and RCSB 49 , 50 databases. CHOPIN 51 and ModBase 52 homology databases were used to identify homology models where crystallographic structures were unavailable.…”

Identification of the Tolfenamic Acid Binding Pocket in PrbP from Liberibacter asiaticus

“…PrbP is a member of CarD_CdnL_TRCF family, a distinct group of proteins that regulate the transcription machinery through interactions with the RNA polymerase (Padmanabhan, 2001 ; Penalver-Mellado et al, 2006 ; Stallings et al, 2009 ; Garcia-Moreno et al, 2010 ; Weiss et al, 2012 ; Gallego-García et al, 2014 ; Bernal-Bernal et al, 2015 ; Gardner et al, 2016 ). Myxococcus xanthus encodes for two members of this family, carD and cdnL.…”