M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation.

Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei (Mm) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation.

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.

How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids.

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.

Diverse molecular mechanisms of transcription regulation by the bacterial alarmone ppGpp.

Bacteria must rapidly detect and respond to stressful environmental conditions. Guanosine tetraphosphate (ppGpp) is a universal stress signal that, in most bacteria, drives the reprograming of transcription at a global level. However, recent studies have revealed that the molecular mechanisms utilized by ppGpp to rewire bacterial transcriptomes are unexpectedly diverse. In Proteobacteria, ppGpp regulates the expression of hundreds of genes by directly binding to two sites on RNA polymerase (RNAP), one in combination with the transcription factor, DksA. Conversely, ppGpp indirectly regulates transcription in Firmicutes by controlling GTP levels. In this case, ppGpp inhibits enzymes that salvage and synthesize GTP, which indirectly represses transcription from rRNA and other promoters that use GTP for initiation. More recently, two different mechanisms of transcription regulation involving the direct binding of transcription factors by ppGpp have been described. First, in Francisella tularensis, ppGpp was shown to modulate the formation of a tripartite transcription factor complex that binds RNAP and activates virulence genes. Second, in Firmicutes, ppGpp allosterically regulates the transcription repressor, PurR, which controls purine biosynthesis genes. The diversity in bacterial ppGpp signaling revealed in these studies suggests the likelihood that additional paradigms in ppGpp-mediated transcription regulation await discovery.

Structural Basis for Virulence Activation of Francisella tularensis.

The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. MglA-SspA assembles with the σ70-associated RNAP holoenzyme (RNAPσ70), forming a virulence-specialized polymerase. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence, but the mechanism is unknown. Here we report FtRNAPσ70-promoter-DNA, FtRNAPσ70-(MglA-SspA)-promoter DNA, and FtRNAPσ70-(MglA-SspA)-ppGpp-PigR-promoter DNA cryo-EM structures. Structural and genetic analyses show MglA-SspA facilitates σ70 binding to DNA to regulate virulence and virulence-enhancing genes. Our Escherichia coli RNAPσ70-homodimeric EcSspA structure suggests this is a general SspA-transcription regulation mechanism. Strikingly, our FtRNAPσ70-(MglA-SspA)-ppGpp-PigR-DNA structure reveals ppGpp binding to MglA-SspA tethers PigR to promoters. PigR in turn recruits FtRNAP αCTDs to DNA UP elements. Thus, these studies unveil a unique mechanism for Ft pathogenesis involving a virulence-specialized RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.

The RRM of the kRNA-editing protein TbRGG2 uses multiple surfaces to bind and remodel RNA.

Kinetoplastid RNA (kRNA) editing takes place in the mitochondria of kinetoplastid protists and creates translatable mRNAs by uridine insertion/deletion. Extensively edited (pan-edited) transcripts contain quadruplex forming guanine stretches, which must be remodeled to promote uridine insertion/deletion. Here we show that the RRM domain of the essential kRNA-editing factor TbRGG2 binds poly(G) and poly(U) RNA and can unfold both. A region C-terminal to the RRM mediates TbRGG2 dimerization, enhancing RNA binding. A RRM-U4 RNA structure reveals a unique RNA-binding mechanism in which the two RRMs of the dimer employ aromatic residues outside the canonical RRM RNA-binding motifs to encase and wrench open the RNA, while backbone atoms specify the uridine bases. Notably, poly(G) RNA is bound via a different binding surface. Thus, these data indicate that TbRGG2 RRM can bind and remodel several RNA substrates suggesting how it might play multiple roles in the kRNA editing process.

The MerR-like protein BldC binds DNA direct repeats as cooperative multimers to regulate Streptomyces development.

Streptomycetes are notable for their complex life cycle and production of most clinically important antibiotics. A key factor that controls entry into development and the onset of antibiotic production is the 68-residue protein, BldC. BldC is a putative DNA-binding protein related to MerR regulators, but lacks coiled-coil dimerization and effector-binding domains characteristic of classical MerR proteins. Hence, the molecular function of the protein has been unclear. Here we show that BldC is indeed a DNA-binding protein and controls a regulon that includes other key developmental regulators. Intriguingly, BldC DNA-binding sites vary significantly in length. Our BldC-DNA structures explain this DNA-binding capability by revealing that BldC utilizes a DNA-binding mode distinct from MerR and other known regulators, involving asymmetric head-to-tail oligomerization on DNA direct repeats that results in dramatic DNA distortion. Notably, BldC-like proteins radiate throughout eubacteria, establishing BldC as the founding member of a new structural family of regulators.