RESUMO
In addition to completing the Watson-Crick nucleobase matching "concept" (big pairs with small, hydrogen bond donors pair with hydrogen bond acceptors), artificially expanded genetic information systems (AEGIS) also challenge DNA polymerases with a complete set of mismatches, including wobble mismatches. Here, we explore wobble mismatches with AEGIS with DNA polymerase 1 from Escherichia coli. Remarkably, we find that the polymerase tolerates an AEGIS:standard wobble that has the same geometry as the G:T wobble that polymerases have evolved to exclude but excludes a wobble geometry that polymerases have never encountered in natural history. These results suggest certain limits to "structural analogy" and "evolutionary guidance" as tools to help synthetic biologists expand DNA alphabets.
Assuntos
Pareamento Incorreto de Bases , DNA Polimerase I/metabolismo , DNA/genética , DNA/metabolismo , Evolução Molecular , Pareamento de Bases , DNA/química , Escherichia coli/enzimologia , Ligação ProteicaRESUMO
MOTIVATION: Despite advances in high-throughput sequencing, marine metagenomic samples remain largely opaque. A typical sample contains billions of microbial organisms from thousands of genomes and quadrillions of DNA base pairs. Its derived metagenomic dataset underrepresents this complexity by orders of magnitude because of the sparseness and shortness of sequencing reads. Read shortness and sequencing errors pose a major challenge to accurate species and functional annotation. This includes distinguishing known from novel species. Often the majority of reads cannot be annotated and thus cannot help our interpretation of the sample. RESULTS: Here, we demonstrate quantitatively how careful assembly of marine metagenomic reads within, but also across, datasets can alleviate this problem. For 10 simulated datasets, each with species complexity modeled on a real counterpart, chimerism remained within the same species for most contigs (97%). For 42 real pyrosequencing ('454') datasets, assembly increased the proportion of annotated reads, and even more so when datasets were pooled, by on average 1.6% (max 6.6%) for species, 9.0% (max 28.7%) for Pfam protein domains and 9.4% (max 22.9%) for PANTHER gene families. Our results outline exciting prospects for data sharing in the metagenomics community. While chimeric sequences should be avoided in other areas of metagenomics (e.g. biodiversity analyses), conservative pooled assembly is advantageous for annotation specificity and sensitivity. Intriguingly, our experiment also found potential prospects for (low-cost) discovery of new species in 'old' data. CONTACT: dgerloff@ffame.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Quimerismo , Conjuntos de Dados como Assunto , Genoma Arqueal , Metagenômica , Anotação de Sequência Molecular/métodos , Análise de Sequência de DNA/métodos , Genoma BacterianoRESUMO
Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not found in natural DNA, but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-ß-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-ß-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.
Assuntos
DNA/química , DNA/síntese química , DNA/genética , Biblioteca Gênica , Células Hep G2 , Humanos , Modelos Moleculares , Reação em Cadeia da PolimeraseRESUMO
Biofilms are dense microbial communities. Although widely distributed and medically important, how biofilm cells interact with one another is poorly understood. Recently, we described a novel process whereby myxobacterial biofilm cells exchange their outer membrane (OM) lipoproteins. For the first time we report here the identification of two host proteins, TraAB, required for transfer. These proteins are predicted to localize in the cell envelope; and TraA encodes a distant PA14 lectin-like domain, a cysteine-rich tandem repeat region, and a putative C-terminal protein sorting tag named MYXO-CTERM, while TraB encodes an OmpA-like domain. Importantly, TraAB are required in donors and recipients, suggesting bidirectional transfer. By use of a lipophilic fluorescent dye, we also discovered that OM lipids are exchanged. Similar to lipoproteins, dye transfer requires TraAB function, gliding motility and a structured biofilm. Importantly, OM exchange was found to regulate swarming and development behaviors, suggesting a new role in cell-cell communication. A working model proposes TraA is a cell surface receptor that mediates cell-cell adhesion for OM fusion, in which lipoproteins/lipids are transferred by lateral diffusion. We further hypothesize that cell contact-dependent exchange helps myxobacteria to coordinate their social behaviors.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Comunicação Celular , Membrana Celular , Metabolismo dos Lipídeos , Myxococcus xanthus/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Adesão Celular/genética , Comunicação Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Metabolismo dos Lipídeos/genética , Proteínas Motores Moleculares/genética , Myxococcus xanthus/citologia , Conformação Proteica , Transporte Proteico/genéticaRESUMO
SUMMARY: The Malaria Genome Exploration Tool (MaGnET) is a software tool enabling intuitive 'exploration-style' visualization of functional genomics data relating to the malaria parasite, Plasmodium falciparum. MaGnET provides innovative integrated graphic displays for different datasets, including genomic location of genes, mRNA expression data, protein-protein interactions and more. Any selection of genes to explore made by the user is easily carried over between the different viewers for different datasets, and can be changed interactively at any point (without returning to a search). AVAILABILITY AND IMPLEMENTATION: Free online use (Java Web Start) or download (Java application archive and MySQL database; requires local MySQL installation) at http://malariagenomeexplorer.org CONTACT: joanna.sharman@ed.ac.uk or dgerloff@ffame.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Genoma de Protozoário , Plasmodium falciparum/genética , Software , Genômica , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
With just four building blocks, low sequence information density, few functional groups, poor control over folding, and difficulties in forming compact folds, natural DNA and RNA have been disappointing platforms from which to evolve receptors, ligands, and catalysts. Accordingly, synthetic biology has created "artificially expanded genetic information systems" (AEGIS) to add nucleotides, functionality, and information density. With the expected improvements seen in AegisBodies and AegisZymes, the task for synthetic biologists shifts to developing for expanded DNA the same analytical tools available to natural DNA. Here we report one of these, an enzyme-assisted sequencing of expanded genetic alphabet (ESEGA) method to sequence six-letter AEGIS DNA. We show how ESEGA analyses this DNA at single base resolution, and applies it to optimized conditions for six-nucleotide PCR, assessing the fidelity of various DNA polymerases, and extending this to AEGIS components with functional groups. This supports the renewed exploitation of expanded DNA alphabets in biotechnology.
Assuntos
DNA , Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA/genética , DNA/metabolismo , Biologia Sintética/métodos , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Análise de Sequência de DNA/métodosRESUMO
Binary subcomplexes in proteins database (BISC) is a new protein-protein interaction (PPI) database linking up the two communities most active in their characterization: structural biology and functional genomics researchers. The BISC resource offers users (i) a structural perspective and related information about binary subcomplexes (i.e. physical direct interactions between proteins) that are either structurally characterized or modellable entries in the main functional genomics PPI databases BioGRID, IntAct and HPRD; (ii) selected web services to further investigate the validity of postulated PPI by inspection of their hypothetical modelled interfaces. Among other uses we envision that this resource can help identify possible false positive PPI in current database records. BISC is freely available at http://bisc.cse.ucsc.edu.
Assuntos
Bases de Dados de Proteínas , Complexos Multiproteicos/química , Bases de Dados Genéticas , Genômica , Modelos Moleculares , Complexos Multiproteicos/genética , Mapeamento de Interação de ProteínasRESUMO
A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022).1.
Assuntos
Bioensaio , Engenharia de Proteínas , Biologia Computacional , Glicosiltransferases/genética , MutaçãoRESUMO
Many efforts have sought to apply laboratory in vitro evolution (LIVE) to natural nucleic acid (NA) scaffolds to directly evolve functional molecules. However, synthetic biology can move beyond natural NA scaffolds to create molecular systems whose libraries are far richer reservoirs of functionality than natural NAs. For example, "artificially expanded genetic information systems" (AEGIS) add up to eight nucleotides to the four found in standard NA. Even in its simplest 6-letter versions, AEGIS adds functional groups, information density, and folding motifs that natural NA libraries lack. To complete this vision, however, tools are needed to sequence molecules that are created by AEGIS LIVE. Previous sequencing approaches, including approaches from our laboratories, exhibited limited performance and lost many sequences in diverse library mixtures. Here, we present a new approach that enzymatically transforms the target AEGIS DNA. With higher transliteration efficiency and fidelity, this Enzyme-Assisted Sequencing of Expanded Genetic Alphabet (ESEGA) approach produces substantially better sequences of 6-letter (AGCTZP) DNA than previous transliteration approaches. Therefore, ESEGA facilitates precise analysis of libraries, allowing 'next-generation deep sequencing' to accurately quantify the sequences of 6-letter DNA molecules at single base resolution. We then applied ESEGA to three tasks: (a) defining optimal conditions to perform 6-nucleotide PCR (b) evaluating the fidelity of 6-nucleotide PCR with various DNA polymerases, and (c) extending that evaluation to AEGIS components functionalized with alkynyl and aromatic groups. No other approach at present has this scope, allowing this work to be the next step towards exploiting the potential of expanded DNA alphabets in biotechnology.
RESUMO
The mechanism of mitotic chromosome condensation is poorly understood, but even less is known about the mechanism of formation of the primary constriction, or centromere. A proteomic analysis of mitotic chromosome scaffolds led to the identification of CENP-V, a novel kinetochore protein related to a bacterial enzyme that detoxifies formaldehyde, a by-product of histone demethylation in eukaryotic cells. Overexpression of CENP-V leads to hypercondensation of pericentromeric heterochromatin, a phenotype that is abolished by mutations in the putative catalytic site. CENP-V depletion in HeLa cells leads to abnormal expansion of the primary constriction of mitotic chromosomes, mislocalization and destabilization of the chromosomal passenger complex (CPC) and alterations in the distribution of H3K9me3 in interphase nucleoplasm. CENP-V-depleted cells suffer defects in chromosome alignment in metaphase, lagging chromosomes in anaphase, failure of cytokinesis and rapid cell death. CENP-V provides a novel link between centromeric chromatin, the primary constriction and the CPC.
Assuntos
Centrômero/metabolismo , Cromossomos Humanos/metabolismo , Citocinese/fisiologia , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona , Cromossomos Humanos/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Heterocromatina/metabolismo , Humanos , Cinetocoros/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Interferência de RNA , Alinhamento de SequênciaRESUMO
Glioblastoma (GBM) is the most aggressive primary brain tumor characterized by infiltrative growth of malignant glioma cells into the surrounding brain parenchyma. In this study, our analysis of GBM patient cohorts revealed a significantly higher expression of Glycosyltransferase 8 domain containing 1 (GLT8D1) compared to normal brain tissue and could be associated with impaired patient survival. Increased in vitro expression of GLT8D1 significantly enhanced migration of two different sphere-forming GBM cell lines. By in silico analysis we predicted the 3D-structure as well as the active site residues of GLT8D1. The introduction of point mutations in the predicted active site reduced its glycosyltransferase activity in vitro and consequently impaired GBM tumor cell migration. Examination of GLT8D1 interaction partners by LC-MS/MS implied proteins associated with cytoskeleton and intracellular transport as potential substrates. In conclusion, we demonstrated that the enzymatic activity of glycosyltransferase GLT8D1 promotes GBM cell migration.
RESUMO
Copy number variations (CNVs) are major causative contributors both in the genesis of genetic diseases and human neoplasias. While "High-Throughput" sequencing technologies are increasingly becoming the primary choice for genomic screening analysis, their ability to efficiently detect CNVs is still heterogeneous and remains to be developed. The aim of this white paper is to provide a guiding framework for the future contributions of ELIXIR's recently established human CNV Community, with implications beyond human disease diagnostics and population genomics. This white paper is the direct result of a strategy meeting that took place in September 2018 in Hinxton (UK) and involved representatives of 11 ELIXIR Nodes. The meeting led to the definition of priority objectives and tasks, to address a wide range of CNV-related challenges ranging from detection and interpretation to sharing and training. Here, we provide suggestions on how to align these tasks within the ELIXIR Platforms strategy, and on how to frame the activities of this new ELIXIR Community in the international context.
Assuntos
Biologia Computacional , Variações do Número de Cópias de DNA , Variações do Número de Cópias de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fuso Acromático , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Divisão Celular , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos , Humanos , Proteínas Inibidoras de Apoptose , Substâncias Macromoleculares , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas de Neoplasias , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , SurvivinaRESUMO
Pfs48/45 and Pfs25 are leading candidates for the development of Plasmodium falciparum transmission blocking vaccines (TBV). Expression of Pfs48/45 in the erythrocytic sexual stages and presentation to the immune system during infection in the human host also makes it ideal for natural boosting. However, it has been challenging to produce a fully folded, functionally active Pfs48/45, using various protein expression platforms. In this study, we demonstrate that full-length Pfs48/45 encoded by DNA plasmids is able to induce significant transmission reducing immune responses. DNA plasmids encoding Pfs48/45 based on native (WT), codon optimized (SYN), or codon optimized and mutated (MUT1 and MUT2), to prevent any asparagine (N)-linked glycosylation were compared with or without intramuscular electroporation (EP). EP significantly enhanced antibody titers and transmission blocking activity elicited by immunization with SYN Pfs48/45 DNA vaccine. Mosquito membrane feeding assays also revealed improved functional immunogenicity of SYN Pfs48/45 (N-glycosylation sites intact) as compared to MUT1 or MUT2 Pfs48/45 DNA plasmids (all N-glycosylation sites mutated). Boosting with recombinant Pfs48/45 protein after immunization with each of the different DNA vaccines resulted in significant boosting of antibody response and improved transmission reducing capabilities of all four DNA vaccines. Finally, immunization with a combination of DNA plasmids (SYN Pfs48/45 and SYN Pfs25) also provides support for the possibility of combining antigens targeting different life cycle stages in the parasite during transmission through mosquitoes.
Assuntos
Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Transmissão de Doença Infecciosa/prevenção & controle , Eletroporação , Feminino , Malária Falciparum/transmissão , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genéticaRESUMO
Antibodies recognizing conformational epitopes in Pfs48/45, an antigen expressed on the surface of Plasmodium falciparum gametes and zygotes, have firmly established Pfs48/45 as a promising transmission blocking vaccine (TBV) candidate. However, it has been difficult to reproducibly express Pfs48/45 in a variety of recombinant expression systems. The goal of our studies was to evaluate functional immunogenicity of Pfs48/45 using DNA vaccine format in rhesus macaques. An additional goal was to ensure that when used in combination with another malarial antigen, specific immunity to both antigens was not compromised. For testing combination vaccines, we employed Pfs25 DNA plasmids that have previously undergone evaluations in rodents and nonhuman primates. Pfs25 is expressed on the surface of parasites after fertilization and is also a lead TBV candidate. DNA plasmids based on codon-optimized sequences of Pfs48/45 and Pfs25 were administered by in vivo electroporation, followed by a final recombinant protein boost. Our studies demonstrate that Pfs48/45 encoded by DNA plasmids is capable of inducing potent transmission blocking antibody responses, and such transmission blocking immune potency of Pfs48/45 was not compromised when tested in combination with Pfs25, These findings provide the evidence in favor of further studies on Pfs48/45 and Pfs25, either alone or in combination with other known malaria vaccine candidates for developing effective vaccines capable of interrupting malaria transmission.
Assuntos
Anticorpos Antiprotozoários/sangue , Glicoproteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Vacinas de DNA/imunologia , Animais , DNA de Protozoário/administração & dosagem , Eletroporação , Feminino , Esquemas de Imunização , Macaca mulatta , Masculino , Glicoproteínas de Membrana/genética , Plasmídeos/administração & dosagem , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Numerous mammalian proteins are constructed from a limited repertoire of module-types. Proteins belonging to the regulators of complement activation family--crucial for ensuring a complement-mediated immune response is targeted against infectious agents--are composed solely of complement control protein (CCP) modules. In the current study, CCP module sequences were grouped to allow selection of the most appropriate experimentally determined structures to serve as templates in an automated large-scale structure modelling procedure. The resulting 135 individual CCP module models, valuable in their own right, are available at the online database http://www.bru.ed.ac.uk/~dinesh/ccp-db.html. Comparisons of surface properties within a particular family of modules should be more informative than sequence alignments alone. A comparison of surface electrostatic features was undertaken for the first 28 CCP modules of complement receptor type 1 (CR1). Assignments to clusters based on surface properties differ from assignments to clusters based on sequences. This observation might reflect adaptive evolution of surface-exposed residues involved in protein-protein interactions. This illustrative example of a multiple surface-comparison was indeed able to pinpoint functional sites in CR1.
Assuntos
Modelos Moleculares , Receptores de Complemento/química , Sequência de Aminoácidos , Animais , Bases de Dados de Proteínas , Humanos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Homologia Estrutural de ProteínaRESUMO
SMC proteins are essential components of three protein complexes that are important for chromosome structure and function. The cohesin complex holds replicated sister chromatids together, whereas the condensin complex has an essential role in mitotic chromosome architecture. Both are involved in interphase genome organization. SMC-containing complexes are large (more than 650 kDa for condensin) and contain long anti-parallel coiled-coils. They are thus difficult subjects for conventional crystallographic and electron cryomicroscopic studies. Here, we have used amino acid-selective cross-linking and mass spectrometry combined with structure prediction to develop a full-length molecular draft three-dimensional structure of the SMC2/SMC4 dimeric backbone of chicken condensin. We assembled homology-based molecular models of the globular heads and hinges with the lengthy coiled-coils modelled in fragments, using numerous high-confidence cross-links and accounting for potential irregularities. Our experiments reveal that isolated condensin complexes can exist with their coiled-coil segments closely apposed to one another along their lengths and define the relative spatial alignment of the two anti-parallel coils. The centres of the coiled-coils can also approach one another closely in situ in mitotic chromosomes. In addition to revealing structural information, our cross-linking data suggest that both H2A and H4 may have roles in condensin interactions with chromatin.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Ligação a DNA/química , Modelos Moleculares , Complexos Multiproteicos/química , Proteínas Nucleares/química , Animais , Linhagem Celular , Galinhas , Cromossomos , Ligação Genética , Histonas/metabolismo , Mitose , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de FusãoRESUMO
The alternative NADH:ubiquinone oxidoreductase (NDH-2) from Escherichia coli is a membrane protein playing a prominent role in respiration by linking the reduction of NADH to the quinone pool. Remote sequence similarity reveals an evolutionary relation between alternative NADH:quinone oxidoreductases and the SCOP-family "FAD/NAD-linked reductases". We have created a structural model for NDH-2 from E. coli through comparative modelling onto a template from this family. Combined analysis of our model and sequence conservation allowed us to include the cofactor FAD and the substrate NADH in atomic detail. Furthermore, we propose the most plausible orientation of NDH-2 relative to the membrane and specify a region of the protein potentially involved in ubiquinone binding.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Respiração Celular/fisiologia , Cobre/metabolismo , Complexo I de Transporte de Elétrons/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , NAD/química , NAD/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ubiquinona/metabolismoRESUMO
UNLABELLED: More than 20% of all protein domains are currently annotated as "domains of unknown function" (DUFs). About 2,700 DUFs are found in bacteria compared with just over 1,500 in eukaryotes. Over 800 DUFs are shared between bacteria and eukaryotes, and about 300 of these are also present in archaea. A total of 2,786 bacterial Pfam domains even occur in animals, including 320 DUFs. Evolutionary conservation suggests that many of these DUFs are important. Here we show that 355 essential proteins in 16 model bacterial species contain 238 DUFs, most of which represent single-domain proteins, clearly establishing the biological essentiality of DUFs. We suggest that experimental research should focus on conserved and essential DUFs (eDUFs) for functional analysis given their important function and wide taxonomic distribution, including bacterial pathogens. IMPORTANCE: The functional units of proteins are domains. Typically, each domain has a distinct structure and function. Genomes encode thousands of domains, and many of the domains have no known function (domains of unknown function [DUFs]). They are often ignored as of little relevance, given that many of them are found in only a few genomes. Here we show that many DUFs are essential DUFs (eDUFs) based on their presence in essential proteins. We also show that eDUFs are often essential even if they are found in relatively few genomes. However, in general, more common DUFs are more often essential than rare DUFs.