Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

Transcription is the principal control point for bacterial gene expression, and it enables a global cellular response to an intracellular or environmental trigger. Transcriptional regulation is orchestrated by transcription factors, which activate or repress transcription of target genes by modulating the activity of RNA polymerase. Dissecting the nature and precise choreography of these interactions is essential for developing a molecular understanding of transcriptional regulation. While the contribution of X-ray crystallography has been invaluable, the ‘resolution revolution’ of cryo-electron microscopy has transformed our structural investigations, enabling large, dynamic and often transient transcription complexes to be resolved that in many cases had resisted crystallisation. In this review, we highlight the impact cryo-electron microscopy has had in gaining a deeper understanding of transcriptional regulation in bacteria. We also provide readers working within the field with an overview of the recent innovations available for cryo-electron microscopy sample preparation and image reconstruction of transcription complexes.

Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism.The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PK TIM+Reg ) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PK TIM+Reg retains a low-level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PK TIM+Reg structure is not significantly altered and is consistent with the wild type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes.