Recruitment of a novel zinc‐bound transcriptional factor by a bacterial HMGA‐type protein is required for regulating multiple processes in Myxococcus xanthus

Abstract

Enhanceosome assembly in eukaryotes often requires high mobility group A (HMGA) proteins. In prokaryotes, the only known transcriptional regulator with HMGA-like physical, structural and DNA-binding properties is Myxococcus xanthus CarD. Here, we report that every CarD-regulated process analysed also requires the product of gene carG, located immediately downstream of and transcriptionally coupled to carD. CarG has the zinc-binding H/C-rich metallopeptidase motif found in archaemetzincins, but with Q replacing a catalytically essential E. CarG, a monomer, binds two zinc atoms, shows no apparent metallopeptidase activity, and its stability in vivo absolutely requires the cysteines. This indicates a strictly structural role for zinc-binding. In vivo CarG localizes to the nucleoid but only if CarD is also present. In vitro CarG shows no DNA-binding but physically interacts with CarD via its N-terminal and not HMGA domain. CarD and CarG thus work as a single, physically linked, transcriptional regulatory unit, and if one exists in a bacterium so does the other. Like zinc-associated eukaryotic transcriptional adaptors in enhanceosome assembly, CarG regulates by interacting not with DNA but with another transcriptional factor.

High-Mobility-Group A-Like CarD Binds to a DNA Site Optimized for Affinity and Position and to RNA Polymerase To Regulate a Light-Inducible Promoter in Myxococcus xanthus

“…Through this domain CarD forms a stable complex with CarG, which is indispensable in every known CarD-dependent process (13,15). CarG has an H/C-rich zinc binding motif similar to archaemetzincin metalloproteases (15,18) but lacks protease activity and does not bind DNA directly (13,15). CarG thus appears to function as a transcriptional adaptor.…”

“…We addressed this in the present study by carrying out a detailed analysis of a site upstream of the light-inducible promoter of the carQRS operon (P QRS ), (Fig. 1B), the only one that has thus far been experimentally demonstrated to be important for both CarD binding and function (6,(13)(14)(15). Expression from P QRS is driven by the RNAP holoenzyme containing CarQ, an alternative factor of the extracytoplasmic function (ECF) family (20).…”

“…CarG thus appears to function as a transcriptional adaptor. Interestingly, the CarD-CarG regulatory complex, composed of an HMGA-like DNA binding factor and a factor that does not bind DNA, is reminiscent of HMGA1a and the p300/CBP adaptor in a eukaryotic enhanceosome, a large nucleoprotein assembly that also includes the basal transcriptional machinery and gene-specific regulators (3,15,19).…”

“…M. xanthus CarD has a C-terminal domain with four AT hooks that resembles human HMGA1a (with three AT hooks) in its physical, structural, and DNA binding properties (Fig. 1A) (13)(14)(15). In contrast to mammalian HMGA, CarD also has a structurally well-defined Nterminal domain of ϳ180 residues which is essential for function and defines the widely distributed CarD_CdnL_TRCF family of bacterial proteins or protein modules that have been implicated in interactions with RNA polymerase (RNAP) (5,(13)(14)(15)(16)(17).…”

“…In contrast to mammalian HMGA, CarD also has a structurally well-defined Nterminal domain of ϳ180 residues which is essential for function and defines the widely distributed CarD_CdnL_TRCF family of bacterial proteins or protein modules that have been implicated in interactions with RNA polymerase (RNAP) (5,(13)(14)(15)(16)(17). Through this domain CarD forms a stable complex with CarG, which is indispensable in every known CarD-dependent process (13,15). CarG has an H/C-rich zinc binding motif similar to archaemetzincin metalloproteases (15,18) but lacks protease activity and does not bind DNA directly (13,15).…”