Structural Insights into RNA Polymerase Recognition and Essential Function of Myxococcus xanthus CdnL

Abstract

CdnL and CarD are two functionally distinct members of the CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-interacting proteins, which co-exist in Myxococcus xanthus. While CarD, found exclusively in myxobacteria, has been implicated in the activity of various extracytoplasmic function (ECF) σ-factors, the function and mode of action of the essential CdnL, whose homologs are widespread among bacteria, remain to be elucidated in M. xanthus. Here, we report the NMR solution structure of CdnL and present a structure-based mutational analysis of its function. An N-terminal five-stranded β-sheet Tudor-like module in the two-domain CdnL mediates binding to RNAP-β, and mutations that disrupt this interaction impair cell growth. The compact CdnL C-terminal domain consists of five α-helices folded as in some tetratricopeptide repeat-like protein-protein interaction domains, and contains a patch of solvent-exposed nonpolar and basic residues, among which a set of basic residues is shown to be crucial for CdnL function. We show that CdnL, but not its loss-of-function mutants, stabilizes formation of transcriptionally competent, open complexes by the primary σA-RNAP holoenzyme at an rRNA promoter in vitro. Consistent with this, CdnL is present at rRNA promoters in vivo. Implication of CdnL in RNAP-σA activity and of CarD in ECF-σ function in M. xanthus exemplifies how two related members within a widespread bacterial protein family have evolved to enable distinct σ-dependent promoter activity.

The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter

“…Previously, CdnL has been shown to directly bind to RNAP and stabilize the open promoter complex required for transcription of rRNA promoters in Mycobacteria and M. xanthus [3,5]. Similarly, Caulobacter CdnL has been shown to bind RNAP and regulate transcription from an rRNA promoter [11].…”

“…CdnL is broadly conserved in bacteria and is best-characterized in Myxococcus xanthus and in Mycobacteria [2][3][4][5][6]. It localizes to promoter regions where the housekeeping sigma factor resides, directly binds to the RNAP β subunit, stabilizes the open promoter complex, and is required for transcription from rRNA promoters [2,3,5]. Recent work in Mycobacterium tuberculosis using mutants of CarD (the CdnL homolog) unable to bind either RNAP or DNA showed that CdnL can act as an activator or repressor of transcription depending on the stability of the RNAP-promoter complex [7].…”

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

“…Its ~140-residue intrinsically unfolded C-terminal DNA-binding domain resembles eukaryotic high-mobility group A (HMGA) proteins and is composed of an acidic segment followed by a region containing the hallmark basic RGRP or AT-hook repeats [ 5 – 7 ]. The CarD N-terminal domain, CarDNt (~180 residues), has defined tertiary structure and interacts with CarG (a 170-residue zinc-associated protein required in all CarD-dependent processes) and with the RNA polymerase (RNAP) β subunit, but not with DNA [ 4 , 5 , 8 – 10 ].…”

“…CdnL is also an essential factor in the few other bacterial species where it has been studied, such as the pathogens Mycobacterium tuberculosis and Borrelia burgdorferi , both of which lack CarD [ 12 , 17 ]. CdnL interacts with RNAP and has been linked to rRNA promoters, where it stabilizes the formation of transcriptionally competent open complexes (RP o ) by RNAP containing the major housekeeping σ, σ A [ 10 , 12 , 18 , 19 ]. Thus, whereas CarD is linked to the action of various ECF-σ factors at their target promoters, CdnL has been implicated in σ A -dependent rRNA promoter activation.…”

Assessment of unconventional antimicrobial compounds for the control of ‘Candidatus Liberibacter asiaticus’, the causative agent of citrus greening disease

“…PrbP is a transcriptional accessory protein belonging to the CarD_CdnL_TRCF family, which regulates gene expression through interactions with RNA polymerase 14,[17][18][19][20][21][22] . Homologs of PrbP in Myxococcus and Mycobacterium are associated with antibiotic resistance, pathogenicity, persistence, oxidative stress response and vegetative growth under starvation conditions 17,19,[23][24][25][26] .…”