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Regulatory networks allow organisms to match adaptive behavior to the complex and dynamic contingencies of their native habitats. Upon a sudden transition to a novel environment, the mismatch between the native behavior and the new niche provides selective

A pervasive question in biological research studying gene regulation, chromatin structure, or genomics is where, and to what extent, does a signal of interest arise genome-wide? This question is addressed using a variety of methods relying on high-throughput sequencing data as their final output, including ChIP-seq for protein-DNA interactions, 1 GapR-seq for measuring supercoiling, 2 and HBD-seq or DRIP-seq for R-loop positioning. 3,4 Current computational methods to calculate genome-wide enrichment of the signal of interest usually do not properly handle the count-based nature of sequencing data, they often do not make use of the local correlation structure of sequencing data, and they do not apply any regularization of enrichment estimates. This can result in unrealistic estimates of the true underlying biological enrichment of interest, unrealistically low estimates of confidence in point estimates of enrichment (or no estimates of confidence at all), unrealistic gyrations in enrichment estimates at very close (<10 bp) genomic loci due to noise inherent in sequencing data, and in a multiple-hypothesis testing problem during interpretation of genome-wide enrichment estimates. We developed a tool called Enricherator to infer genome-wide enrichments from sequencing count data. Enricherator uses the variational Bayes algorithm to fit a generalized linear model to sequencing count data and to sample from the approximate posterior distribution of enrichment estimates (https://github.com/jwschroeder3/enricherator). Enrichments inferred by Enricherator more precisely identify known binding sites in cases where low coverage between binding sites leads to false-positive peak calls in these noisy regions of the genome; these benefits extend to published datasets., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: P.L. Freddolino is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for Scientific Reports and EcoSal Plus; neither organization was involved in the editorial review or the decision to publish this article. P.L. Freddolino is on the Scientific Advisory Board and is a Consultant for CircNova, Inc; CircNova provided no financial support for this work, and was not involved in any way in the performance of the research, manuscript preparation, editorial review, or decision to publish this article. J.W. Schroeder declares that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recent research has indicated the presence of heterochromatin-like regions of extended protein occupancy and transcriptional silencing of bacterial genomes. We utilized an integrative approach to track chromatin structure and transcription in E. coli K-12 across a wide range of nutrient conditions. In the process, we identified multiple loci which act similarly to facultative heterochromatin in eukaryotes, normally silenced but permitting expression of genes under specific conditions. We also found a strong enrichment of small regulatory RNAs (sRNAs) among the set of differentially expressed transcripts during nutrient stress. Using a newly developed bioinformatic pipeline, the transcription factors regulating sRNA expression were bioinformatically predicted, with experimental follow-up revealing novel relationships for 36 sRNA-transcription factors candidates. Direct regulation of sRNA expression was confirmed by mutational analysis for five sRNAs of metabolic interest: IsrB, CsrB and CsrC, GcvB, and GadY. Our integrative analysis thus reveals additional layers of complexity in the nutrient stress response in E. coli and provides a framework for revealing similar poorly understood regulatory logic in other organisms., Competing Interests: CONFLICT OF INTEREST The authors declare no conflicts of interest.

The sequence-specific RNA-binding protein Pumilio (Pum) controls Drosophila development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we use knockdown and knockout approaches coupled with RNA-seq to measure the impact of Pum on the transcriptome of Drosophila cells in culture. We also use an improved RNA coimmunoprecipitation method to identify Pum-bound mRNAs in Drosophila embryos. Integration of these data sets with the locations of Pum-binding motifs across the transcriptome reveals novel direct Pum target genes involved in neural, muscle, wing, and germ cell development and in cellular proliferation. These genes include components of Wnt, TGF-β, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pum-mediated repression, and observe concordant regulation of Pum:CCR4-NOT target mRNAs. Computational modeling reveals that Pum binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pum-mediated repression are not influenced by the content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pum regulatory network and mechanisms and the parameters that influence the efficacy of Pum-mediated regulation., (© 2024 Haugen et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Leveraging iterative alignment search through genomic and metagenome sequence databases, we report the DeepMSA2 pipeline for uniform protein single- and multichain multiple-sequence alignment (MSA) construction. Large-scale benchmarks show that DeepMSA2 MSAs can remarkably increase the accuracy of protein tertiary and quaternary structure predictions compared with current state-of-the-art methods. An integrated pipeline with DeepMSA2 participated in the most recent CASP15 experiment and created complex structural models with considerably higher quality than the AlphaFold2-Multimer server (v.2.2.0). Detailed data analyses show that the major advantage of DeepMSA2 lies in its balanced alignment search and effective model selection, and in the power of integrating huge metagenomics databases. These results demonstrate a new avenue to improve deep learning protein structure prediction through advanced MSA construction and provide additional evidence that optimization of input information to deep learning-based structure prediction methods must be considered with as much care as the design of the predictor itself., (© 2024. The Author(s).)

With the progress of structural biology, the Protein Data Bank (PDB) has witnessed rapid accumulation of experimentally solved protein structures. Since many structures are determined with purification and crystallization additives that are unrelated to a protein's in vivo function, it is nontrivial to identify the subset of protein-ligand interactions that are biologically relevant. We developed the BioLiP2 database (https://zhanggroup.org/BioLiP) to extract biologically relevant protein-ligand interactions from the PDB database. BioLiP2 assesses the functional relevance of the ligands by geometric rules and experimental literature validations. The ligand binding information is further enriched with other function annotations, including Enzyme Commission numbers, Gene Ontology terms, catalytic sites, and binding affinities collected from other databases and a manual literature survey. Compared to its predecessor BioLiP, BioLiP2 offers significantly greater coverage of nucleic acid-protein interactions, and interactions involving large complexes that are unavailable in PDB format. BioLiP2 also integrates cutting-edge structural alignment algorithms with state-of-the-art structure prediction techniques, which for the first time enables composite protein structure and sequence-based searching and significantly enhances the usefulness of the database in structure-based function annotations. With these new developments, BioLiP2 will continue to be an important and comprehensive database for docking, virtual screening, and structure-based protein function analyses., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We report the results of the "UM-TBM" and "Zheng" groups in CASP15 for protein monomer and complex structure prediction. These prediction sets were obtained using the D-I-TASSER and DMFold-Multimer algorithms, respectively. For monomer structure prediction, D-I-TASSER introduced four new features during CASP15: (i) a multiple sequence alignment (MSA) generation protocol that combines multi-source MSA searching and a structural modeling-based MSA ranker; (ii) attention-network based spatial restraints; (iii) a multi-domain module containing domain partition and arrangement for domain-level templates and spatial restraints; (iv) an optimized I-TASSER-based folding simulation system for full-length model creation guided by a combination of deep learning restraints, threading alignments, and knowledge-based potentials. For 47 free modeling targets in CASP15, the final models predicted by D-I-TASSER showed average TM-score 19% higher than the standard AlphaFold2 program. We thus showed that traditional Monte Carlo-based folding simulations, when appropriately coupled with deep learning algorithms, can generate models with improved accuracy over end-to-end deep learning methods alone. For protein complex structure prediction, DMFold-Multimer generated models by integrating a new MSA generation algorithm (DeepMSA2) with the end-to-end modeling module from AlphaFold2-Multimer. For the 38 complex targets, DMFold-Multimer generated models with an average TM-score of 0.83 and Interface Contact Score of 0.60, both significantly higher than those of competing complex prediction tools. Our analyses on complexes highlighted the critical role played by MSA generating, ranking, and pairing in protein complex structure prediction. We also discuss future room for improvement in the areas of viral protein modeling and complex model ranking., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Misfolded endoplasmic reticulum (ER) proteins are degraded through a process called ER-associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identify several regions required for different Hrd1 functions. Most surprisingly, we find two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we define roles for disordered regions between structural elements that are required for Hrd1 autoubiquitination and substrate interaction. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the ER membrane., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Modeling and simulation of small molecules such as drugs and biological cofactors have been both a major focus of computational chemistry for decades and a growing need among computational biophysicists who seek to investigate the interaction of different types of ligands with biomolecules. Of particular interest in this regard are quantum mechanical (QM) calculations that are used to more accurately describe such small molecules, which can be of heterogeneous structures and chemistry, either in purely QM calculations or in hybrid QM/molecular mechanics (MM) simulations. QM programs are also used to develop MM force field parameters for small molecules to be used along with established force fields for biomolecules in classical simulations. With this growing need in mind, here we report a set of software tools developed and closely integrated within the broadly used molecular visualization/analysis program, VMD, that allow the user to construct, modify, and parametrize small molecules and prepare them for QM, hybrid QM/MM, or classical simulations. The tools also provide interactive analysis and visualization capabilities in an easy-to-use and integrated environment. In this paper, we briefly report on these tools and their major features and capabilities, along with examples of how they can facilitate molecular research in computational biophysics that might be otherwise prohibitively complex.

RNA:DNA hybrids compromise replication fork progression and genome integrity in all cells. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate strongly in noncoding RNAs and 5'-UTRs of coding sequences. For Δ rnhB , hybrids accumulate preferentially in untranslated regions and early in coding sequences. We show that hybrid accumulation is particularly sensitive to gene expression in Δ rnhC cells. DNA replication in Δ rnhC cells is disrupted, leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts cause mutagenesis and shape genome organization.

Feast-famine response proteins are a widely conserved class of global regulators in prokaryotes, the most highly studied of which is the Escherichia coli leucine-responsive regulatory protein (Lrp). Lrp senses the environmental nutrition status and subsequently regulates up to one-third of the genes in E. coli, either directly or indirectly. Lrp exists predominantly as octamers and hexadecamers (16mers), where leucine is believed to shift the equilibrium toward the octameric state. In this study, we analyzed the effects of three oligomerization state mutants of Lrp in terms of their ability to bind to DNA and regulate gene expression in response to exogenous leucine. We find that oligomerization beyond dimers is required for Lrp's regulatory activity and that, contrary to previous speculation, exogenous leucine modulates Lrp activity at its target promoters exclusively by inhibiting Lrp binding to DNA. We also show evidence that Lrp binding bridges DNA over length scales of multiple kilobases, revealing a new range of mechanisms for Lrp-mediated transcriptional regulation. IMPORTANCE Leucine-responsive regulatory protein (Lrp) is one of the most impactful regulators in E. coli and other bacteria. Lrp senses nutrient conditions and responds by controlling strategies for virulence, cellular motility, and nutrient acquisition. Despite its importance and being evolutionarily highly conserved across bacteria and archaea, several mysteries remain regarding Lrp, including how it actually responds to leucine to change its regulation of targets. Previous studies have led to the hypothesis that Lrp switches between two states, an octamer (8 Lrp molecules together) and a hexadecamer (16 Lrp molecules together), upon exposure to leucine; these are referred to as different oligomerization states. Here, we show that contrary to previous expectations, it is Lrp's propensity to bind DNA, rather than its oligomerization state, that is directly affected by leucine in the cell's environment. Our new understanding of Lrp activity will aid in identifying and disrupting pathways used by bacteria to cause disease.

Diet profoundly influences brain physiology, but how metabolic information is transmuted into neural activity and behavior changes remains elusive. Here, we show that the metabolic enzyme O-GlcNAc Transferase (OGT) moonlights on the chromatin of the D. melanogaster gustatory neurons to instruct changes in chromatin accessibility and transcription that underlie sensory adaptations to a high-sugar diet. OGT works synergistically with the Mitogen Activated Kinase/Extracellular signal Regulated Kinase (MAPK/ERK) rolled and its effector stripe (also known as EGR2 or Krox20) to integrate activity information. OGT also cooperates with the epigenetic silencer Polycomb Repressive Complex 2.1 (PRC2.1) to decrease chromatin accessibility and repress transcription in the high-sugar diet. This integration of nutritional and activity information changes the taste neurons' responses to sugar and the flies' ability to sense sweetness. Our findings reveal how nutrigenomic signaling generates neural activity and behavior in response to dietary changes in the sensory neurons., Competing Interests: HS, AV, DW, RW, PF, MD No competing interests declared, (© 2023, Sung, Vaziri et al.)

Thymidine starvation causes rapid cell death. This enigmatic process known as thymineless death (TLD) is the underlying killing mechanism of diverse antimicrobial and antineoplastic drugs. Despite decades of investigation, we still lack a mechanistic understanding of the causal sequence of events that culminate in TLD. Here, we used a diverse set of unbiased approaches to systematically determine the genetic and regulatory underpinnings of TLD in Escherichia coli. In addition to discovering novel genes in previously implicated pathways, our studies revealed a critical and previously unknown role for intracellular acidification in TLD. We observed that a decrease in cytoplasmic pH is a robust early event in TLD across different genetic backgrounds. Furthermore, we show that acidification is a causal event in the death process, as chemical and genetic perturbations that increase intracellular pH substantially reduce killing. We also observe a decrease in intracellular pH in response to exposure to the antibiotic gentamicin, suggesting that intracellular acidification may be a common mechanistic step in the bactericidal effects of other antibiotics., Competing Interests: The authors have declared that no competing interests exist.

Recent experiments have shown that in addition to control by cis regulatory elements, the local chromosomal context of a gene also has a profound impact on its transcription. Although this chromosome-position dependent expression variation has been empirically mapped at high-resolution, the underlying causes of the variation have not been elucidated. Here, we demonstrate that 1 kb of flanking, non-coding synthetic sequences with a low frequency of guanosine and cytosine (GC) can dramatically reduce reporter expression compared to neutral and high GC-content flanks in Escherichia coli. Natural and artificial genetic context can have a similarly strong effect on reporter expression, regardless of cell growth phase or medium. Despite the strong reduction in the maximal expression level from the fully-induced reporter, low GC synthetic flanks do not affect the time required to reach the maximal expression level after induction. Overall, we demonstrate key determinants of transcriptional propensity that appear to act as tunable modulators of transcription, independent of regulatory sequences such as the promoter. These findings provide insight into the regulation of naturally occurring genes and an independent control for optimizing expression of synthetic biology constructs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Gene Ontology (GO) has been widely used to annotate functions of genes and gene products. Here, we proposed a new method, TripletGO, to deduce GO terms of protein-coding and non-coding genes, through the integration of four complementary pipelines built on transcript expression profile, genetic sequence alignment, protein sequence alignment, and naïve probability. TripletGO was tested on a large set of 5754 genes from 8 species (human, mouse, Arabidopsis, rat, fly, budding yeast, fission yeast, and nematoda) and 2433 proteins with available expression data from the third Critical Assessment of Protein Function Annotation challenge (CAFA3). Experimental results show that TripletGO achieves function annotation accuracy significantly beyond the current state-of-the-art approaches. Detailed analyses show that the major advantage of TripletGO lies in the coupling of a new triplet network-based profiling method with the feature space mapping technique, which can accurately recognize function patterns from transcript expression profiles. Meanwhile, the combination of multiple complementary models, especially those from transcript expression and protein-level alignments, improves the coverage and accuracy of the final GO annotation results. The standalone package and an online server of TripletGO are freely available at https://zhanggroup.org/TripletGO/., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Thioesterases play a critical role in metabolism, membrane biosynthesis, and overall homeostasis for all domains of life. In this present study, we characterize a putative thioesterase from Escherichia coli MG1655 and define its role as a cytosolic enzyme. Building on structure-guided functional predictions, we show that YigI is a medium- to long-chain acyl-CoA thioesterase that is involved in the degradation of conjugated linoleic acid (CLA) in vivo , showing overlapping specificity with two previously defined E. coli thioesterases TesB and FadM. We then bioinformatically identify the regulatory relationships that induce YigI expression, which include: an acidic environment, high oxygen availability, and exposure to aminoglycosides. Our findings define a role for YigI and shed light on why the E. coli genome harbors numerous thioesterases with closely related functions. IMPORTANCE Previous research has shown that long chain acyl-CoA thioesterases are needed for E. coli to grow in the presence of carbon sources such as conjugated linoleic acid, but that E. coli must possess at least one such enzyme that had not previously been characterized. Building off structure-guided function predictions, we showed that the poorly annotated protein YigI is indeed the previously unidentified third acyl CoA thioesterase. We found that the three potentially overlapping acyl-CoA thioesterases appear to be induced by nonoverlapping conditions and use that information as a starting point for identifying the precise reactions catalyzed by each such thioesterase, which is an important prerequisite for their industrial application and for more accurate metabolic modeling of E. coli.

HP1 proteins traverse a complex and crowded chromatin landscape to bind with low affinity but high specificity to histone H3K9 methylation (H3K9me) and form transcriptionally inactive genomic compartments called heterochromatin. Here, we visualize single-molecule dynamics of an HP1 homolog, the fission yeast Swi6, in its native chromatin environment. By tracking single Swi6 molecules, we identify mobility states that map to discrete biochemical intermediates. Using Swi6 mutants that perturb H3K9me recognition, oligomerization, or nucleic acid binding, we determine how each biochemical property affects protein dynamics. We estimate that Swi6 recognizes H3K9me3 with ~94-fold specificity relative to unmodified nucleosomes in living cells. While nucleic acid binding competes with Swi6 oligomerization, as few as four tandem chromodomains can overcome these inhibitory effects to facilitate Swi6 localization at heterochromatin formation sites. Our studies indicate that HP1 oligomerization is essential to form dynamic, higher-order complexes that outcompete nucleic acid binding to enable specific H3K9me recognition.

Deep learning techniques have significantly advanced the field of protein structure prediction. LOMETS3 (https://zhanglab.ccmb.med.umich.edu/LOMETS/) is a new generation meta-server approach to template-based protein structure prediction and function annotation, which integrates newly developed deep learning threading methods. For the first time, we have extended LOMETS3 to handle multi-domain proteins and to construct full-length models with gradient-based optimizations. Starting from a FASTA-formatted sequence, LOMETS3 performs four steps of domain boundary prediction, domain-level template identification, full-length template/model assembly and structure-based function prediction. The output of LOMETS3 contains (i) top-ranked templates from LOMETS3 and its component threading programs, (ii) up to 5 full-length structure models constructed by L-BFGS (limited-memory Broyden-Fletcher-Goldfarb-Shanno algorithm) optimization, (iii) the 10 closest Protein Data Bank (PDB) structures to the target, (iv) structure-based functional predictions, (v) domain partition and assembly results, and (vi) the domain-level threading results, including items (i)-(iii) for each identified domain. LOMETS3 was tested in large-scale benchmarks and the blind CASP14 (14th Critical Assessment of Structure Prediction) experiment, where the overall template recognition and function prediction accuracy is significantly beyond its predecessors and other state-of-the-art threading approaches, especially for hard targets without homologous templates in the PDB. Based on the improved developments, LOMETS3 should help significantly advance the capability of broader biomedical community for template-based protein structure and function modelling., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Proteome-wide identification of protein-protein interactions is a formidable task which has yet to be sufficiently addressed by experimental methodologies. Many computational methods have been developed to predict proteome-wide interaction networks, but few leverage both the sensitivity of structural information and the wide availability of sequence data. We present PEPPI, a pipeline which integrates structural similarity, sequence similarity, functional association data, and machine learning-based classification through a naïve Bayesian classifier model to accurately predict protein-protein interactions at a proteomic scale. Through benchmarking against a set of 798 ground truth interactions and an equal number of non-interactions, we have found that PEPPI attains 4.5% higher AUROC than the best of other state-of-the-art methods. As a proteomic-scale application, PEPPI was applied to model the interactions which occur between SARS-CoV-2 and human host cells during coronavirus infection, where 403 high-confidence interactions were identified with predictions covering 73% of a gold standard dataset from PSICQUIC and demonstrating significant complementarity with the most recent high-throughput experiments. PEPPI is available both as a webserver and in a standalone version and should be a powerful and generally applicable tool for computational screening of protein-protein interactions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

At the time of this writing, December 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlaps the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple RBD mutations have risen to dominance. Nonadditive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape, particularly the risk of vaccine escape, is crucial. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the wild type and Delta, Gamma, and Omicron variants. Overall, epistasis at the RBD interface appears to be limited, and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2 interaction, whereas in Gamma, epistasis more substantially destabilizes NAb interaction. Despite bearing many more RBD mutations, the epistatic landscape of Omicron closely resembles that of Gamma. Thus, although Omicron poses new risks not observed with Delta, structural constraints on the RBD appear to hamper continued evolution toward more complete vaccine escape. The modest ensemble of mutations relative to the wild type that are currently known to reduce vaccine efficacy is likely to contain the majority of all possible escape mutations for future variants, predicting the continued efficacy of the existing vaccines. IMPORTANCE Emergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild-type SARS-CoV-2 virus as well as the Delta, Gamma, and Omicron variants. The efficacy of the existing vaccines against Omicron could be substantially reduced relative to the wild type, and the potential for vaccine escape is of grave concern. Our results suggest that although Omicron poses new evolutionary risks not observed for Delta, structural constraints on the RBD make continued evolution toward more complete vaccine escape from either Delta or Omicron unlikely. The modest set of escape-enhancing mutations already identified for the wild type likely include the majority of all possible mutations with this effect.

Cryo-electron microscopy (cryo-EM) has become a leading approach for protein structure determination, but it remains challenging to accurately model atomic structures with cryo-EM density maps. We propose a hybrid method, CR-I-TASSER (cryo-EM iterative threading assembly refinement), which integrates deep neural-network learning with I-TASSER assembly simulations for automated cryo-EM structure determination. The method is benchmarked on 778 proteins with simulated and experimental density maps, where CR-I-TASSER constructs models with a correct fold (template modeling (TM) score >0.5) for 643 targets that is 64% higher than the best of some other de novo and refinement-based approaches on high-resolution data samples. Detailed data analyses showed that the main advantage of CR-I-TASSER lies in the deep learning-based Cα position prediction, which significantly improves the threading template quality and therefore boosts the accuracy of final models through optimized fragment assembly simulations. These results demonstrate a new avenue to determine cryo-EM protein structures with high accuracy and robustness covering various target types and density map resolutions., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

There is increasing evidence that prokaryotes maintain chromosome structure, which in turn impacts gene expression. We recently characterized densely occupied, multi-kilobase regions in the E. coli genome that are transcriptionally silent, similar to eukaryotic heterochromatin. These extended protein occupancy domains (EPODs) span genomic regions containing genes encoding metabolic pathways as well as parasitic elements such as prophages. Here, we investigate the contributions of nucleoid-associated proteins (NAPs) to the structuring of these domains, by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and B. subtilis. We identify key NAPs contributing to the silencing of specific EPODs, whose deletion opens a chromosomal region for RNA polymerase binding at genes contained within that region. We show that changes in E. coli EPODs facilitate an extra layer of transcriptional regulation, which prepares cells for exposure to exotic carbon sources. Furthermore, we distinguish novel xenogeneic silencing roles for the NAPs Fis and Hfq, with the presence of at least one being essential for cell viability in the presence of domesticated prophages. Our findings reveal previously unrecognized mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements., (© 2021 The Authors.)

Heterochromatin is most often associated with eukaryotic organisms. Yet, bacteria also contain areas with densely protein-occupied chromatin that appear to silence gene expression. One nucleoid-associated silencing factor is the conserved protein Hfq. Although seemingly nonspecific in its DNA binding properties, Hfq is strongly enriched at AT-rich DNA regions, characteristic of prophages and mobile genetic elements. Here, we demonstrate that polyphosphate (polyP), an ancient and highly conserved polyanion, is essential for the site-specific DNA binding properties of Hfq in bacteria. Absence of polyP markedly alters the DNA binding profile of Hfq, causes unsolicited prophage and transposon mobilization, and increases mutagenesis rates and DNA damage–induced cell death. In vitro reconstitution of the system revealed that Hfq and polyP interact with AT-rich DNA sequences and form phase-separated condensates, a process that is mediated by the intrinsically disordered C-terminal extensions of Hfq. We propose that polyP serves as a newly identified driver of heterochromatin formation in bacteria.

Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst -/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst -/- mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst -/- mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Since the discovery of the Escherichia coli leucine-responsive regulatory protein (Lrp) almost 50 years ago, hundreds of Lrp homologs have been discovered, occurring in 45% of sequenced bacteria and almost all sequenced archaea. Lrp-like proteins are often referred to as the feast/famine regulatory proteins (FFRPs), reflecting their common regulatory roles. Acting as either global or local transcriptional regulators, FFRPs detect the environmental nutritional status by sensing small effector molecules (usually amino acids) and regulate the expression of genes involved in metabolism, virulence, motility, nutrient transport, stress tolerance, and antibiotic resistance to implement appropriate behaviors for the specific ecological niche of each organism. Despite FFRPs' complexity, a significant role in gene regulation, and prevalence throughout prokaryotes, the last comprehensive review on this family of proteins was published about a decade ago. In this review, we integrate recent notable findings regarding E. coli Lrp and other FFRPs across bacteria and archaea with previous observations to synthesize a more complete view on the mechanistic details and biological roles of this ancient class of transcription factors.

Genome architecture has proven to be critical in determining gene regulation across almost all domains of life. While many of the key components and mechanisms of eukaryotic genome organization have been described, the interplay between bacterial DNA organization and gene regulation is only now being fully appreciated. An increasing pool of evidence has demonstrated that the bacterial chromosome can reasonably be thought of as chromatin, and that bacterial chromosomes contain transcriptionally silent and transcriptionally active regions analogous to heterochromatin and euchromatin, respectively. The roles played by histones in eukaryotic systems appear to be shared across a range of nucleoid-associated proteins (NAPs) in bacteria, which function to compact, structure, and regulate large portions of bacterial chromosomes. The broad range of extant NAPs, and the extent to which they differ from species to species, has raised additional challenges in identifying and characterizing their roles in all but a handful of model bacteria. Here we review the regulatory roles played by NAPs in several well-studied bacteria and use the resulting state of knowledge to provide a working definition for NAPs, based on their function, binding pattern, and expression levels. We present a screening procedure which can be applied to any species for which transcriptomic data are available. Finally, we note that NAPs tend to play two major regulatory roles - xenogeneic silencers and developmental regulators - and that many unrecognized potential NAPs exist in each bacterial species examined.

Bacterial small RNAs (sRNAs) play a vital role in pathogenesis by enabling rapid, efficient networks of gene attenuation during infection. In recent decades, there has been a surge in the number of proposed and biochemically-confirmed sRNAs in both Gram-positive and Gram-negative pathogens. However, limited homology, network complexity, and condition specificity of sRNA has stunted complete characterization of the activity and regulation of these RNA regulators. To streamline the discovery of the expression of sRNAs, and their post-transcriptional activities, we propose an integrative in vivo data-mining approach that couples DNA protein occupancy, RNA-seq, and RNA accessibility data with motif identification and target prediction algorithms. We benchmark the approach against a subset of well-characterized E. coli sRNAs for which a degree of in vivo transcriptional regulation and post-transcriptional activity has been previously reported, finding support for known regulation in a large proportion of this sRNA set. We showcase the abilities of our method to expand understanding of sRNA RseX, a known envelope stress-linked sRNA for which a cellular role has been elusive due to a lack of native expression detection. Using the presented approach, we identify a small set of putative RseX regulators and targets for experimental investigation. These findings have allowed us to confirm native RseX expression under conditions that eliminate H-NS repression as well as uncover a post-transcriptional role of RseX in fimbrial regulation. Beyond RseX, we uncover 163 putative regulatory DNA-binding protein sites, corresponding to regulation of 62 sRNAs, that could lead to new understanding of sRNA transcription regulation. For 32 sRNAs, we also propose a subset of top targets filtered by engagement of regions that exhibit binding site accessibility behavior in vivo . We broadly anticipate that the proposed approach will be useful for sRNA-reliant network characterization in bacteria. Such investigations under pathogenesis-relevant environmental conditions will enable us to deduce complex rapid-regulation schemes that support infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mihailovic, Ekdahl, Chen, Leistra, Li, González Martínez, Law, Ejindu, Massé, Freddolino and Contreras.)

Free-living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein-DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using an optimized version of in vivo protein occupancy display technology (in vivo protein occupancy display-high resolution, IPOD-HR) and present a pilot application to Escherichia coli. We observe that the E. coli protein-DNA interactome organizes into 2 distinct prototypic features: (1) highly dynamic condition-dependent transcription factor (TF) occupancy; and (2) robust kilobase scale occupancy by nucleoid factors, forming silencing domains analogous to eukaryotic heterochromatin. We show that occupancy dynamics across a range of conditions can rapidly reveal the global transcriptional regulatory organization of a bacterium. Beyond discovery of previously hidden regulatory logic, we show that these observations can be utilized to computationally determine sequence specificity models for the majority of active TFs. Our study demonstrates that global observations of protein occupancy combined with statistical inference can rapidly and systematically reveal the transcriptional regulatory and structural features of a bacterial genome. This capacity is particularly crucial for non-model bacteria that are not amenable to routine genetic manipulation., Competing Interests: The authors have declared that no competing interests exist.

When the JCVI-syn3.0 genome was designed and implemented in 2016 as the minimal genome of a free-living organism, approximately one-third of the 438 protein-coding genes had no known function. Subsequent refinement into JCVI-syn3A led to inclusion of 16 additional protein-coding genes, including several unknown functions, resulting in an improved growth phenotype. Here, we seek to unveil the biological roles and protein-protein interaction (PPI) networks for these poorly characterized proteins using state-of-the-art deep learning contact-assisted structure prediction, followed by structure-based annotation of functions and PPI predictions. Our pipeline is able to confidently assign functions for many previously unannotated proteins such as putative vitamin transporters, which suggest the importance of nutrient uptake even in a minimized genome. Remarkably, despite the artificial selection of genes in the minimal syn3 genome, our reconstructed PPI network still shows a power law distribution of node degrees typical of naturally evolved bacterial PPI networks. Making use of our framework for combined structure/function/interaction modeling, we are able to identify both fundamental aspects of network biology that are retained in a minimal proteome and additional essential functions not yet recognized among the poorly annotated components of the syn3.0 and syn3A proteomes.

Diets rich in sugar, salt, and fat alter taste perception and food preference, contributing to obesity and metabolic disorders, but the molecular mechanisms through which this occurs are unknown. Here, we show that in response to a high sugar diet, the epigenetic regulator Polycomb Repressive Complex 2.1 (PRC2.1) persistently reprograms the sensory neurons of Drosophila melanogaster flies to reduce sweet sensation and promote obesity. In animals fed high sugar, the binding of PRC2.1 to the chromatin of the sweet gustatory neurons is redistributed to repress a developmental transcriptional network that modulates the responsiveness of these cells to sweet stimuli, reducing sweet sensation. Half of these transcriptional changes persist despite returning the animals to a control diet, causing a permanent decrease in sweet taste. Our results uncover a new epigenetic mechanism that, in response to the dietary environment, regulates neural plasticity and feeding behavior to promote obesity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

The human PUF-family proteins, PUM1 and PUM2, posttranscriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3'-UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady-state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a "rulebook" of key contextual features that differentiate functional versus nonfunctional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins., (© 2020 Wolfe et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We find that NOCT protein is differentially expressed and processed in a cell and tissue type-specific manner to control its localization to the cytoplasm or mitochondrial exterior or interior. The N terminus of NOCT is necessary and sufficient to confer import and processing in the mitochondria. We measured the impact of cytoplasmic NOCT on the transcriptome and observed that it affects mRNA levels of hundreds of genes that are significantly enriched in osteoblast, neuronal, and mitochondrial functions. Recent biochemical data indicate that NOCT dephosphorylates NADP(H) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in a manner specified by its location in cells., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Abshire et al.)

Motivation: Many protein function databases are built on automated or semi-automated curations and can contain various annotation errors. The correction of such misannotations is critical to improving the accuracy and reliability of the databases., Results: We proposed a new approach to detect potentially incorrect Gene Ontology (GO) annotations by comparing the ratio of annotation rates (RAR) for the same GO term across different taxonomic groups, where those with a relatively low RAR usually correspond to incorrect annotations. As an illustration, we applied the approach to 20 commonly studied species in two recent UniProt-GOA releases and identified 250 potential misannotations in the 2018-11-6 release, where only 25% of them were corrected in the 2019-6-3 release. Importantly, 56% of the misannotations are 'Inferred from Biological aspect of Ancestor (IBA)' which is in contradiction with previous observations that attributed misannotations mainly to 'Inferred from Sequence or structural Similarity (ISS)', probably reflecting an error source shift due to the new developments of function annotation databases. The results demonstrated a simple but efficient misannotation detection approach that is useful for large-scale comparative protein function studies., Availability and Implementation: https://zhanglab.ccmb.med.umich.edu/RAR., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

It has been more than a decade since the last comprehensive review of the phase-variable uropathogen-associated pyelonephritis-associated pilus ( pap ) genetic switch. Since then, important data have come to light, including additional factors that regulate pap expression, better characterization of H-NS regulation, the structure of the Lrp octamer in complex with pap regulatory DNA, the temperature-insensitive phenotype of a mutant lacking the acetyltransferase RimJ, evidence that key components of the regulatory machinery are acetylated, and new insights into the role of DNA binding by key regulators in shaping both the physical structure and regulatory state of the papI and papBA promoters. This review revisits pap , integrating these newer observations with older ones to produce a new model for the concerted behavior of this virulence-regulatory region., (Copyright © 2020 American Society for Microbiology.)

Pumilio is an RNA-binding protein that represses a network of mRNAs to control embryogenesis, stem cell fate, fertility and neurological functions in Drosophila. We sought to identify the mechanism of Pumilio-mediated repression and find that it accelerates degradation of target mRNAs, mediated by three N-terminal Repression Domains (RDs), which are unique to Pumilio orthologs. We show that the repressive activities of the Pumilio RDs depend on specific subunits of the Ccr4-Not (CNOT) deadenylase complex. Depletion of Pop2, Not1, Not2, or Not3 subunits alleviates Pumilio RD-mediated repression of protein expression and mRNA decay, whereas depletion of other CNOT components had little or no effect. Moreover, the catalytic activity of Pop2 deadenylase is important for Pumilio RD activity. Further, we show that the Pumilio RDs directly bind to the CNOT complex. We also report that the decapping enzyme, Dcp2, participates in repression by the N-terminus of Pumilio. These results support a model wherein Pumilio utilizes CNOT deadenylase and decapping complexes to accelerate destruction of target mRNAs. Because the N-terminal RDs are conserved in mammalian Pumilio orthologs, the results of this work broadly enhance our understanding of Pumilio function and roles in diseases including cancer, neurodegeneration and epilepsy., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function., Results: Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory., Conclusion: We conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

Metabolites are active controllers of cellular physiology, but their role in complex behaviors is less clear. Here we report metabolic changes that occur during the transition between hunger and satiety in Drosophila melanogaster. To analyze these data in the context of fruit fly metabolic networks, we developed Flyscape, an open-access tool. We show that in response to eating, metabolic profiles change in quick, but distinct ways in the heads and bodies. Consumption of a high sugar diet dulls the metabolic and behavioral differences between the fasted and fed state, and reshapes the way nutrients are utilized upon eating. Specifically, we found that high dietary sugar increases TCA cycle activity, alters neurochemicals, and depletes 1-carbon metabolism and brain health metabolites N-acetyl-aspartate and kynurenine. Together, our work identifies the metabolic transitions that occur during hunger and satiation, and provides a platform to study the role of metabolites and diet in complex behavior.

CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Recent studies find that sugar tastes less intense to humans with obesity, but whether this sensory change is a cause or a consequence of obesity is unclear. To tackle this question, we study the effects of a high sugar diet on sweet taste sensation and feeding behavior in Drosophila melanogaster. On this diet, fruit flies have lower taste responses to sweet stimuli, overconsume food, and develop obesity. Excess dietary sugar, but not obesity or dietary sweetness alone, caused taste deficits and overeating via the cell-autonomous action of the sugar sensor O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in the sweet-sensing neurons. Correcting taste deficits by manipulating the excitability of the sweet gustatory neurons or the levels of OGT protected animals from diet-induced obesity. Our work demonstrates that the reshaping of sweet taste sensation by excess dietary sugar drives obesity and highlights the role of glucose metabolism in neural activity and behavior., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent studies on targeted gene integrations in bacteria have demonstrated that chromosomal location can substantially affect a gene's expression level. However, these studies have only provided information on a small number of sites. To measure position effects on transcriptional propensity at high resolution across the genome, we built and analyzed a library of over 144,000 genome-integrated, standardized reporters in a single mixed population of Escherichia coli. We observed more than 20-fold variations in transcriptional propensity across the genome when the length of the chromosome was binned into broad 4 kbp regions; greater variability was observed over smaller regions. Our data reveal peaks of high transcriptional propensity centered on ribosomal RNA operons and core metabolic genes, while prophages and mobile genetic elements were enriched in less transcribable regions. In total, our work supports the hypothesis that E. coli has evolved gene-independent mechanisms for regulating expression from specific regions of its genome., (Copyright © 2019 Elsevier Inc. All rights reserved.)