ABSTRACT
In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory cis-element DNA and a cryo-EM structure of GlnR-TAC which comprises Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved ß flap, σAR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.
Subject(s)
Actinobacteria , Actinobacteria/genetics , Actinobacteria/metabolism , Transcriptional Activation/genetics , Bacterial Proteins/metabolism , Trans-Activators/metabolism , Promoter Regions, Genetic/genetics , Gene Expression Regulation, BacterialABSTRACT
The MerR-family transcription factors (TFs) are a large group of bacterial proteins responding to cellular metal ions and multiple antibiotics by binding within central RNA polymerase-binding regions of a promoter. While most TFs alter transcription through protein-protein interactions, MerR TFs are capable of reshaping promoter DNA. To address the question of which mechanism prevails, we determined two cryo-EM structures of transcription activation complexes (TAC) comprising Escherichia coli CueR (a prototype MerR TF), RNAP holoenzyme and promoter DNA. The structures reveal that this TF promotes productive promoter-polymerase association without canonical protein-protein contacts seen between other activator proteins and RNAP. Instead, CueR realigns the key promoter elements in the transcription activation complex by clamp-like protein-DNA interactions: these induce four distinct kinks that ultimately position the -10 element for formation of the transcription bubble. These structural and biochemical results provide strong support for the DNA distortion paradigm of allosteric transcriptional control by MerR TFs.
Subject(s)
Bacterial Proteins/chemistry , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , DNA-Directed RNA Polymerases/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Trans-Activators/chemistry , Allosteric Regulation , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Pairing , Base Sequence , Binding Sites , Cryoelectron Microscopy , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Molecular , Nucleic Acid Conformation , Promoter Regions, Genetic , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Sequence Alignment , Sequence Homology, Amino Acid , Trans-Activators/genetics , Trans-Activators/metabolism , Transcriptional ActivationABSTRACT
σS is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σS-mediated transcription requires a σS-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an Escherichia coli transcription activation complex (E. coli Crl-TAC) comprising E. coli σS-RNA polymerase (σS-RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σS (σS2) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σS2 to promote the assembly of the σS-RNAP holoenzyme and also to facilitate formation of an RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.
