The second model hypothesizes that BAM's assembly of RcsF into outer membrane proteins (OMPs) is disrupted by specific stresses on the outer membrane (OM) or periplasmic gel (PG), ultimately triggering Rcs activation by the unassembled RcsF. These models are not fundamentally incompatible. To illuminate the stress sensing mechanism, we subject these two models to rigorous critical evaluation. Within the Cpx sensor, NlpE, you find both an N-terminal domain (NTD) and a C-terminal domain (CTD). A fault in the lipoprotein transport system causes NlpE to be retained within the inner membrane, consequently instigating the Cpx response. Signaling pathways depend on the NlpE NTD, but not the NlpE CTD; meanwhile, OM-anchored NlpE recognizes hydrophobic surface contact, the NlpE CTD proving essential to this process.
The active and inactive forms of the Escherichia coli cAMP receptor protein (CRP), a model bacterial transcription factor, are contrasted to generate a paradigm elucidating the cAMP-driven activation of CRP. Studies of CRP and CRP*, a collection of CRP mutants lacking cAMP, provide biochemical support for the observed paradigm. The cAMP affinity of CRP is influenced by two factors: (i) the performance of the cAMP pocket and (ii) the equilibrium of the apo-CRP form. The mechanism by which these two factors determine the cAMP affinity and specificity of CRP and CRP* mutants is analyzed. Current insights into, and the gaps in our knowledge concerning, CRP-DNA interactions are also documented. Future consideration of several key CRP issues is underscored by this review's conclusion.
Predicting the future, as Yogi Berra famously stated, is a particularly daunting task, and it's certainly a concern for anyone attempting a manuscript of the present time. The study of Z-DNA's history highlights the fallibility of earlier assumptions regarding its biological implications, ranging from the overly optimistic claims of its proponents, whose predictions have yet to be validated experimentally, to the skepticism of the broader scientific community, who may have dismissed the research as misguided, given the technological limitations of the time. While early predictions might be interpreted favorably, they still did not encompass the biological roles we now understand for Z-DNA and Z-RNA. Using a combination of approaches, especially those derived from human and mouse genetic studies, in conjunction with biochemical and biophysical characterization of the Z family of proteins, the field experienced remarkable progress. The initial success related to the p150 Z isoform of ADAR1 (adenosine deaminase RNA specific), with the cell death research community later providing insights into the functional aspects of ZBP1 (Z-DNA-binding protein 1). As the substitution of basic clockwork with precise instruments changed expectations in navigation, the finding of the roles nature has assigned to structures like Z-DNA has permanently altered our view of the genome's function. The recent breakthroughs have arisen from an integration of better methodologies and advanced analytical approaches. In this article, the methods integral to these remarkable discoveries will be elucidated, and particular areas for future method development that hold promise for further advancements in our knowledge will be highlighted.
Double-stranded RNA editing by adenosine deaminase acting on RNA 1 (ADAR1) is crucial in modulating cellular responses to various RNA sources, both internal and external, via the conversion of adenosine to inosine. ADAR1, the principal enzyme for A-to-I RNA editing in humans, predominantly works on Alu elements, a type of short interspersed nuclear element, which are abundant within the introns and 3' untranslated regions of RNA. The ADAR1 protein exists in two isoforms, p110 (110 kDa) and p150 (150 kDa), whose expression is usually linked; disrupting this linkage has revealed that the p150 isoform's ability to modify targets surpasses that of the p110 isoform. Several approaches for detecting ADAR1-related modifications have been created, and we describe a specific method for identifying edit sites connected to particular ADAR1 isoforms.
Eukaryotic cells actively monitor for viral infections by identifying conserved virus-derived molecular structures, known as pathogen-associated molecular patterns (PAMPs). The presence of PAMPs is usually associated with the replication of viruses, and they are not typically observed in uninfected cells. Double-stranded RNA (dsRNA), a prevalent pathogen-associated molecular pattern (PAMP), is created by most, if not every RNA virus, and by a considerable number of DNA viruses as well. Regarding dsRNA conformation, the molecule can be found in a right-handed (A-RNA) or a left-handed (Z-RNA) double-helical structure. The cytosolic pattern recognition receptors (PRRs), RIG-I-like receptor MDA-5 and the dsRNA-dependent protein kinase PKR, are stimulated by the presence of A-RNA. Z-form nucleic acid binding protein 1 (ZBP1) and the p150 subunit of adenosine deaminase RNA-specific 1 (ADAR1), which are examples of Z domain-containing pattern recognition receptors (PRRs), are responsible for detecting Z-RNA. AD5584 It has been recently shown that Z-RNA is created during orthomyxovirus infections, including those caused by influenza A virus, and serves as an activating ligand for the ZBP1 protein. This chapter details our method for identifying Z-RNA within influenza A virus (IAV)-affected cells. Moreover, this procedure reveals the potential for identifying Z-RNA, a byproduct of vaccinia virus infection, as well as Z-DNA induced by a small-molecule DNA intercalator.
The canonical B or A conformation, while prevalent in DNA and RNA helices, is not exclusive; the flexible conformational landscape of nucleic acids enables exploration of numerous higher-energy states. In the realm of nucleic acid structures, the Z-conformation is exceptional due to its left-handed helical arrangement and its zigzagging backbone. Z-DNA/RNA binding domains, specifically Z domains, are the mechanism by which the Z-conformation is recognized and stabilized. Our recent findings underscore that diverse RNA types can adopt partial Z-conformations, called A-Z junctions, upon interaction with Z-DNA; this structural adoption could depend on both the specific RNA sequence and the surrounding context. This chapter describes general methods for characterizing the interaction of Z domains with RNAs forming A-Z junctions, to ascertain the binding affinity and stoichiometry of these interactions, and further assess the extent and localization of Z-RNA formation.
The physical characteristics of molecules and their reaction mechanisms can be readily studied through direct visualization of the target molecules. Atomic force microscopy (AFM) allows for the direct, nanometer-scale imaging of biomolecules, upholding physiological conditions. In conjunction with DNA origami, the exact positioning of target molecules within a meticulously designed nanostructure is now possible, and single-molecule detection has become a reality. High-speed atomic force microscopy (HS-AFM), integrated with DNA origami, facilitates the visualization of biomolecular dynamic movements, achieving sub-second time resolution for analysis. AD5584 High-resolution atomic force microscopy (HS-AFM), in conjunction with a DNA origami setup, enables the direct visualization of dsDNA rotation during the B-Z transition. These target-oriented observation systems allow for the detailed, real-time analysis of DNA structural changes with molecular precision.
Due to their effects on DNA metabolic processes—including replication, transcription, and genome maintenance—alternative DNA structures, such as Z-DNA, which differ from the canonical B-DNA double helix, have recently received considerable attention. Non-B-DNA-forming sequences can act as a catalyst for genetic instability, a critical factor in the development and evolution of diseases. In different species, Z-DNA can instigate a range of genetic instability events, and several distinct assays have been created to identify the Z-DNA-induced DNA strand breaks and mutagenesis in prokaryotic and eukaryotic systems. Within this chapter, several methodologies are introduced, such as Z-DNA-induced mutation screening and the identification of Z-DNA-induced strand breaks in mammalian cells, yeast, and mammalian cell extracts. Better understanding of the mechanisms behind Z-DNA's connection to genetic instability will emerge from the data collected through these assays in a variety of eukaryotic model systems.
We present a deep learning approach leveraging convolutional and recurrent neural networks to synthesize information from DNA sequences, nucleotide physical, chemical, and structural properties, alongside omics data encompassing histone modifications, methylation, chromatin accessibility, and transcription factor binding sites, and incorporating insights from other available next-generation sequencing experiments. We detail the process of employing a trained model for comprehensive whole-genome annotation of Z-DNA regions, culminating in a feature importance analysis to pinpoint crucial determinants of functional Z-DNA regions.
Left-handed Z-DNA's initial detection was greeted with fervent excitement, signifying a dramatic departure from the standard right-handed double helical configuration of typical B-DNA. This chapter explores the ZHUNT program's computational approach to mapping Z-DNA in genomic sequences, focusing on the rigorous thermodynamic modeling of the B-Z transition. The discussion commences with a succinct overview of the structural distinctions between Z-DNA and B-DNA, specifically concentrating on the characteristics relevant to the B-to-Z transition and the junction where a left-handed DNA helix connects with a right-handed one. AD5584 The statistical mechanics (SM) analysis of the zipper model is subsequently employed to decipher the cooperative B-Z transition, and it accurately replicates the behavior of naturally occurring sequences that undergo the B-Z transition in response to negative supercoiling. We detail the ZHUNT algorithm, its validation, previous applications in genomic and phylogenomic studies, and provide information on accessing the online application.