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G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit 1 . To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR–G-protein complex. By monitoring the transitions of the stimulatory G s protein in complex with the β 2 -adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein–receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events. Time-resolved cryo-EM is used to capture structural transitions during G-protein activation stimulated by a G-protein-coupled receptor.

Protein–RNA and protein–DNA complexes play critical roles in biology. Despite considerable recent advances in protein structure prediction, the prediction of the structures of protein–nucleic acid complexes without homology to known complexes is a largely unsolved problem. Here we extend the RoseTTAFold machine learning protein-structure-prediction approach to additionally predict nucleic acid and protein–nucleic acid complexes. We develop a single trained network, RoseTTAFoldNA, that rapidly produces three-dimensional structure models with confidence estimates for protein–DNA and protein–RNA complexes. Here we show that confident predictions have considerably higher accuracy than current state-of-the-art methods. RoseTTAFoldNA should be broadly useful for modeling the structure of naturally occurring protein–nucleic acid complexes, and for designing sequence-specific RNA and DNA-binding proteins. RoseTTAFoldNA extends the RoseTTAFold2 platform to predict the structures of protein–DNA and protein–RNA complexes.

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal ‘fastener’ helix retains PAF and is followed by a ‘latch’ that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation.Cryo-EM elucidation of a fully reconstituted Pol II–DSF–PAF1–SPT6 elongation complex defines the position of PAF1 subunit RTF1 and reveals contacts with the Pol II bridge helix that may allosterically stimulate transcription elongation.

Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo. Some transcription factors have been proposed to functionally interact with RNA to facilitate proper regulation of gene expression. Here the authors demonstrate that human Sox2 interact directly and with high affinity to RNAs through its HMG DNA-binding domain.

Key Points Bacterial proteins can be damaged by oxidants that are present in the environment. Cys and Met residues are easily oxidized. Bacterial cells have a range of proteins that repair oxidized proteins. Thioredoxins (Trxs) and glutaredoxins (Grxs) repair oxidized cysteine residues. Methionine sulfoxide reductases (Msrs) repair oxidized methionine residues. Antioxidant defences are present in the bacterial cytoplasm and in extracytoplasmic compartments. Oxidative damage can have a devastating effect on the structure and activity of proteins, leading to cell death. This Review discusses how bacteria repair oxidized proteins and highlights the importance of these repair systems in physiology and virulence. Oxidative damage can have a devastating effect on the structure and activity of proteins, and may even lead to cell death. The sulfur-containing amino acids cysteine and methionine are particularly susceptible to reactive oxygen species (ROS) and reactive chlorine species (RCS), which can damage proteins. In this Review, we discuss our current understanding of the reducing systems that enable bacteria to repair oxidatively damaged cysteine and methionine residues in the cytoplasm and in the bacterial cell envelope. We highlight the importance of these repair systems in bacterial physiology and virulence, and we discuss several examples of proteins that become activated by oxidation and help bacteria to respond to oxidative stress.

Despite decades of surveillance and pharmaceutical and non-pharmaceutical interventions, seasonal influenza viruses continue to cause epidemics around the world each year. The key process underlying these recurrent epidemics is the evolution of the viruses to escape the immunity that is induced by prior infection or vaccination. Although we are beginning to understand the processes that underlie the evolutionary dynamics of seasonal influenza viruses, the timing and nature of emergence of new virus strains remain mostly unpredictable. In this Review, we discuss recent advances in understanding the molecular determinants of influenza virus immune escape, sources of evolutionary selection pressure, population dynamics of influenza viruses and prospects for better influenza virus control.

Key Points Specific protein-based and riboswitch-based metal sensors monitor the intracellular levels of metal ions and regulate the expression of pathways for uptake, storage and efflux, as well as alternative enzymes that use a different metal or non-metal cofactor. Transcription factors often mediate graded responses in which different genes are regulated at different levels of signal. Metal ions are required for growth, with cellular concentrations of Zn( II ), Mn( II ) and Fe between 0.4–1 mM under sufficient conditions. Metals are present in metalloenzymes, which are stored in membrane or protein compartments, and are present in a low-molecular-weight labile pool. Inhibition of bacterial growth due to metal limitation often occurs as a result of the failure of metal-dependent enzymes. Inhibition of bacterial growth due to metal intoxication can involve the production of harmful reactive oxygen species and/or the incorrect metallation of enzymes that are involved in key metabolic pathways. The host immune system has evolved to take advantage of both metal limitation ('nutritional immunity') and metal intoxication as methods of responding to infection. Metal limitation and intoxication are evolutionarily conserved mechanisms that are used by protozoa and higher eukaryotes to kill bacteria. In this Review, Chandrangsu et al . discuss recent insights into metalloregulatory systems that are used by bacteria and how they respond to metal limitation and intoxication, as well as how these systems influence host–pathogen interactions. Metal ions are essential for many reactions, but excess metals can be toxic. In bacteria, metal limitation activates pathways that are involved in the import and mobilization of metals, whereas excess metals induce efflux and storage. In this Review, we highlight recent insights into metal homeostasis, including protein-based and RNA-based sensors that interact directly with metals or metal-containing cofactors. The resulting transcriptional response to metal stress takes place in a stepwise manner and is reinforced by post-transcriptional regulatory systems. Metal limitation and intoxication by the host are evolutionarily ancient strategies for limiting bacterial growth. The details of the resulting growth restriction are beginning to be understood and seem to be organism-specific.

Mitochondrial apoptosis is strictly controlled by BCL-2 family proteins through a subtle network of protein interactions. The tumor suppressor protein p53 triggers transcription-independent apoptosis through direct interactions with BCL-2 family proteins, but the molecular mechanism is not well understood. In this study, we present three crystal structures of p53-DBD in complex with the anti-apoptotic protein BCL-2 at resolutions of 2.3–2.7 Å. The structures show that two loops of p53-DBD penetrate directly into the BH3-binding pocket of BCL-2. Structure-based mutations at the interface impair the p53/BCL-2 interaction. Specifically, the binding sites for p53 and the pro-apoptotic protein Bax in the BCL-2 pocket are mostly identical. In addition, formation of the p53/BCL-2 complex is negatively correlated with the formation of BCL-2 complexes with pro-apoptotic BCL-2 family members. Defects in the p53/BCL-2 interaction attenuate p53-mediated cell apoptosis. Overall, our study provides a structural basis for the interaction between p53 and BCL-2, and suggests a molecular mechanism by which p53 regulates transcription-independent apoptosis by antagonizing the interaction of BCL-2 with pro-apoptotic BCL-2 family members. The human tumor suppressor p53 interacts with the BCL-2 family proteins to regulate apoptosis. Here, the authors solve the structures of p53 in complex with the antiapoptotic protein BCL-2 and suggest a mechanism by which p53 promotes apoptosis by competitively antagonizing the interaction of BCL-2 with pro-apoptotic BCL-2 family proteins.

Genetic and genomic techniques have proven incredibly powerful for identifying and studying molecular players implicated in the epigenetic regulation of DNA-templated processes such as transcription. However, achieving a mechanistic understanding of how these molecules interact with chromatin to elicit a functional output is non-trivial, owing to the tremendous complexity of the biochemical networks involved. Advances in protein engineering have enabled the reconstitution of ‘designer’ chromatin containing customized post-translational modification patterns, which, when used in conjunction with sophisticated biochemical and biophysical methods, allow many mechanistic questions to be addressed. In this Review, we discuss how such tools complement established ‘omics’ techniques to answer fundamental questions on chromatin regulation, focusing on chromatin mark establishment and protein–chromatin interactions.In this Review, Hananya et al. discuss how designer chromatin containing specific patterns of post-translational modifications is being used to study the mechanisms underlying the epigenetic regulation of gene expression.

BRAF plays an indispensable role in activating the MEK/ERK pathway to drive tumorigenesis. Receptor tyrosine kinase and RAS-mediated BRAF activation have been extensively characterized, however, it remains undefined how BRAF function is fine-tuned by stimuli other than growth factors. Here, we report that in response to proinflammatory cytokines, BRAF is subjected to lysine 27-linked poly-ubiquitination in melanoma cells by the ITCH ubiquitin E3 ligase. Lysine 27-linked ubiquitination of BRAF recruits PP2A to antagonize the S365 phosphorylation and disrupts the inhibitory interaction with 14–3–3, leading to sustained BRAF activation and subsequent elevation of the MEK/ERK signaling. Physiologically, proinflammatory cytokines activate ITCH to maintain BRAF activity and to promote proliferation and invasion of melanoma cells, whereas the ubiquitination-deficient BRAF mutant displays compromised kinase activity and reduced tumorigenicity. Collectively, our study reveals a pivotal role for ITCH-mediated BRAF ubiquitination in coordinating the signals between cytokines and the MAPK pathway activation in melanoma cells. BRAF drives MEK/ERK activation to facilitate tumorigenesis. Here, the authors show that in response to pro-inflammatory cytokines, ITCH mediates a non-proteolytic ubiquitination and activation of BRAF, which in turn sustains MEK/ERK signaling to facilitate melanoma cell growth.