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Cellular translation surveillance rescues ribosomes that stall on problematic mRNAs. During translation surveillance, endonucleolytic cleavage of the problematic mRNA is a critical step in rescuing stalled ribosomes. Here we identify NONU-1 as a factor required for translation surveillance pathways including no-go and nonstop mRNA decay. We show that (1) NONU-1 reduces nonstop and no-go mRNA levels; (2) NONU-1 contains an Smr RNase domain required for mRNA decay; (3) the domain architecture and catalytic residues of NONU-1 are conserved throughout metazoans and eukaryotes, respectively; and (4) NONU-1 is required for the formation of mRNA cleavage fragments in the vicinity of stalled ribosomes. We extend our results in C. elegans to homologous factors in S. cerevisiae, showing the evolutionarily conserved function of NONU-1. Our work establishes the identity of a factor critical to translation surveillance and will inform mechanistic studies at the intersection of translation and mRNA decay.

BackgroundN-terminal domains of BVU_4064 and BF1687 proteins from Bacteroides vulgatus and Bacteroides fragilis respectively are members of the Pfam family PF12985 (DUF3869). Proteins containing a domain from this family can be found in most Bacteroides species and, in large numbers, in all human gut microbiome samples. Both BVU_4064 and BF1687 proteins have a consensus lipobox motif implying they are anchored to the membrane, but their functions are otherwise unknown. The C-terminal half of BVU_4064 is assigned to protein family PF12986 (DUF3870); the equivalent part of BF1687 was unclassified.ResultsCrystal structures of both BVU_4064 and BF1687 proteins, solved at the JCSG center, show strikingly similar three-dimensional structures. The main difference between the two is that the two domains in the BVU_4064 protein are connected by a short linker, as opposed to a longer insertion made of 4 helices placed linearly along with a strand that is added to the C-terminal domain in the BF1687 protein. The N-terminal domain in both proteins, corresponding to the PF12985 (DUF3869) domain is a β-sandwich with pre-albumin-like fold, found in many proteins belonging to the Transthyretin clan of Pfam. The structures of C-terminal domains of both proteins, corresponding to the PF12986 (DUF3870) domain in BVU_4064 protein and an unclassified domain in the BF1687 protein, show significant structural similarity to bacterial pore-forming toxins. A helix in this domain is in an analogous position to a loop connecting the second and third strands in the toxin structures, where this loop is implicated to play a role in the toxin insertion into the host cell membrane. The same helix also points to the groove between the N- and C-terminal domains that are loosely held together by hydrophobic and hydrogen bond interactions. The presence of several conserved residues in this region together with these structural determinants could make it a functionally important region in these proteins.ConclusionsStructural analysis of BVU_4064 and BF1687 points to possible roles in mediating multiple interactions on the cell-surface/extracellular matrix. In particular the N-terminal domain could be involved in adhesive interactions, the C-terminal domain and the inter-domain groove in lipid or carbohydrate interactions.

BackgroundCA_C2195 from Clostridium acetobutylicum is a protein of unknown function. Sequence analysis predicted that part of the protein contained a metallopeptidase-related domain. There are over 200 homologs of similar size in large sequence databases such as UniProt, with pairwise sequence identities in the range of ~40-60%. CA_C2195 was chosen for crystal structure determination for structure-based function annotation of novel protein sequence space.ResultsThe structure confirmed that CA_C2195 contained an N-terminal metallopeptidase-like domain. The structure revealed two extra domains: an α+β domain inserted in the metallopeptidase-like domain and a C-terminal circularly permuted winged-helix-turn-helix domain.ConclusionsBased on our sequence and structural analyses using the crystal structure of CA_C2195 we provide a view into the possible functions of the protein. From contextual information from gene-neighborhood analysis, we propose that rather than being a peptidase, CA_C2195 and its homologs might play a role in biosynthesis of a modified cell-surface carbohydrate in conjunction with several sugar-modification enzymes. These results provide the groundwork for the experimental verification of the function.

BackgroundA novel highly conserved protein domain, DUF162 [Pfam: PF02589], can be mapped to two proteins: LutB and LutC. Both proteins are encoded by a highly conserved LutABC operon, which has been implicated in lactate utilization in bacteria. Based on our analysis of its sequence, structure, and recent experimental evidence reported by other groups, we hereby redefine DUF162 as the LUD domain family.ResultsJCSG solved the first crystal structure [PDB:2G40] from the LUD domain family: LutC protein, encoded by ORF DR_1909, of Deinococcus radiodurans. LutC shares features with domains in the functionally diverse ISOCOT superfamily. We have observed that the LUD domain has an increased abundance in the human gut microbiome.ConclusionsWe propose a model for the substrate and cofactor binding and regulation in LUD domain. The significance of LUD-containing proteins in the human gut microbiome, and the implication of lactate metabolism in the radiation-resistance of Deinococcus radiodurans are discussed.

BackgroundEvery genome contains a large number of uncharacterized proteins that may encode entirely novel biological systems. Many of these uncharacterized proteins fall into related sequence families. By applying sequence and structural analysis we hope to provide insight into novel biology.ResultsWe analyze a previously uncharacterized Pfam protein family called DUF4424 [Pfam:PF14415]. The recently solved three-dimensional structure of the protein lpg2210 from Legionella pneumophila provides the first structural information pertaining to this family. This protein additionally includes the first representative structure of another Pfam family called the YARHG domain [Pfam:PF13308]. The Pfam family DUF4424 adopts a 19-stranded beta-sandwich fold that shows similarity to the N-terminal domain of leukotriene A-4 hydrolase. The YARHG domain forms an all-helical domain at the C-terminus. Structure analysis allows us to recognize distant similarities between the DUF4424 domain and individual domains of M1 aminopeptidases and tricorn proteases, which form massive proteasome-like capsids in both archaea and bacteria.ConclusionsBased on our analyses we hypothesize that the DUF4424 domain may have a role in forming large, multi-component enzyme complexes. We suggest that the YARGH domain may play a role in binding a moiety in proximity with peptidoglycan, such as a hydrophobic outer membrane lipid or lipopolysaccharide.

The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01 Å using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation.

SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein-protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0).

A study report which suggests that at the evolutionary dynamics of the bacterial transcriptional regulatory network might exhibit distinct patterns at shorter phylogenetic scales is presented.

The article analyzes data from protist genomes to study the evolution of eukaryotic regulatory systems. Comparative genomics reconstructs and diversifies the regulatory systems.

Lipid modification of the N-terminal Cys residue (N-acyl-S-diacylglyceryl-Cys) has been found to be an essential, ubiquitous, and unique bacterial posttranslational modification. Such a modification allows anchoring of even highly hydrophilic proteins to the membrane which carry out a variety of functions important for bacteria, including pathogenesis. Hence, being able to identify such proteins is of great value. To this end, we have created a comprehensive database of bacterial lipoproteins, called DOLOP, which contains information and links to molecular details for about 278 distinct lipoproteins and predicted lipoproteins from 234 completely sequenced bacterial genomes. The website also features a tool that applies a predictive algorithm to identify the presence or absence of the lipoprotein signal sequence in a user-given sequence. The experimentally verified lipoproteins have been classified into different functional classes and more importantly functional domain assignments using hidden Markov models from the SUPERFAMILY database that have been provided for the predicted lipoproteins. Other features include the following: primary sequence analysis, signal sequence analysis, and search facility and information exchange facility to allow researchers to exchange results on newly characterized lipoproteins. The website, along with additional information on the biosynthetic pathway, statistics on predicted lipoproteins, and related figures, is available at http://www.mrc-lmb.cam.ac.uk/genomes/dolop/.

Researchers found genetic differences between two marine planktonic archaea, one from the Antarctic and one from the temperate North Pacific. The Antarctic species produced several proteins that have never before been detected in the Archaea, including an RNA-binding protein in the bacterial cold shock family and a zinc finger protein.

The genomic analysis in Deinococcus radiodurans reveal that the bacterium has developed protective features to resist extreme radiation and desiccation resistance in terms of specific superfamily of enzymes and proteins. Data also point out to the occurrence of numerous nucleotide repeats in the genome that are involved in stress resistance.

Eugene V. Koonin (1) Kira S. Makarova (1,2) L. Aravind (1) Key Words phylogenetic analysis, gene acquisition, gene displacement, adaptation, orthologs * Abstract Comparative analysis of bacterial, archaeal, and eukaryotic [...]

Comparative genomics and its impact on the understanding of evolution are discussed. In 2100 ancient families of orthologs identified by comparing 21 archaeal, bacterial, and eukaryotic genomes, only 80 are found in each organism. Yet genome comparisons show the great unity of all living things. Most of the universal families have core components of the translation and transcription mechanisms. Researchers now know that there is no true understanding of the connection between genome and phenotype in an organism. This is in some sense the central theme of biology. It can be solved only by using an experimental program based on comparative-genomic results.

A detailed analysis of the sequences of the principal domains and proteins involved in programmed cell death was performed using the gapped BLAST program and the iterative program, PSI-BLAST. Results reveal that programmed cell death, or apoptosis, began with the recruitment of ancient domains that performed other functions in unicellular organisms. The last common ancestor of the multicellular crown-group eukaryotes were revealed to have a primitive prototype of the cell-death signaling systems. The evolution was accompanied by a major diversification of the cell-death process.

A hitherto-undescribed (HD) superfamily of predicted phosphohydrolases was characterized. This superfamily of enzymes was related to metalloenzymes, specifically the eukaryotic cyclic-nucleotide phosphodiesterases. These enzymes were predicted to be capable of phosphohydrolase activities and were involved in signal transduction and nucleic acid metabolism. Three motifs were identified in the enzymes: I, II and V. Motif I and V possessed histidine and aspartate residues, respectively, while motif II contained the HD signature.

A previously undetected domain with a Cx[Cx.sub.n]CxH pattern of predicted zinc-chelating residues was identified in a variety of prokaryotic and eukaryotic proteins. These include bacterial ATPases of the SWI2/SNF2 family, plant MuDR transposases and transposase-derived Far1 nuclear proteins, and vertebrate MEK kinase-1. This domain was designated SWIM after SWI2/SNF2 and MuDR, and is predicted to have DNA-binding and protein-protein interaction functions in different contexts.

A newly discovered protein motif called a BED-finger is described. It is found in cellular regulatory factors and transposes, indicating that mobile elements may have been involved in the evolution of chromosomal proteins.

Researchers describe a DNA-binding protein with structural similarity to the scaffold attachment factors A and B. It could be involved in gene transcription, splicing, DNA repair, and apoptotic destruction of chromatin.

Systematic studies on eukaryotic proteins associated with ribonucleic acid (RNA) processing have identified a number of distinct types of RNA-interacting domains that have elucidated the specificity if RNA-protein interactions and enabled the prediction of new RNA-bind proteins by sequence analysis. A study describes a previously undetected domain that is present in a number of RNA-associated proteins and in type D retroviral polyproteins. The characteristic of this domain is its glycine-rich signature, hence its name G-patch.

The steroidogenic acute regulatory protein (StAR)-related lipid-transfer (START) domain has the ability to bind lipids in signalling proteins that have diverse architectures. Web-based resources were used to identify these and other signalling domains from sequence information. It was demonstrated that these sequences are members of a homologous domain family that also includes bovine phosphatidylcholine-transfer protein (PC-T). Studies have shown that residues that contribute to the PC-T lipid-binding site are well conserved among representative START domains.

The name HORMA domain, which is an abbreviation for Hop1p, Rev7p and MAD2, was suggested to denote the sequence conservation observed in DNA repair, chromosome synapsis and mitotic checkpoints. The different alignment of the HORMA domains demonstrated that a number of conserved motifs are incorporated. Moreover, two amino acid residues seemed constant. One is a glutamate residue located at the center of the domain, while the other is an arginine residue at the N-terminal region of the domain.

The P-type ATPases belong to a superfamily of hydrolases whose structure is typified by the L-2-haloacid dehalogenase (HAD) 7,8 from Pseudomonas sp. The catalytic mechanism of P-type ATPases and HAD 7,8 are similar, providing clues to the evolution of these enzymes. The catalytic domain of P-type ATPases has been found to have the structural fold of haloacid dehalogenases. Thus, P-type ATPases might have evolved by fusion of a transmembrane protein with a HAD superfamily phosphatase domain.

Systematic sequencing of bacterial, archaeal and eukaryotic genomes has enabled the delineation of entire sets of family members in not a few sequenced genomes and also the establishment of relationships among these proteins. A novel family of predicted phosphoesterases includes the Drosophila prune gene which, when mutated, produces a lethal genetic combination of a gene encoding nucleoside diphosphate kinase. This DHH family is only one among several families of phosphoiesterases with a broad spectrum of substrates.

This review discusses the unusual features of Plasmodium genome as exhibited by the highly A+T rich rodent malarial parasites. The genome is characterized by displaying large amount of low complexity inserts in their protein globular domains, differences in its transcription machinery, its DNA repair system with vulnerability for mutations, and 'animal-like' adhesion molecules on the cell surface.

A previously uncharacterized 110-125 residue-long domain was identified both in the physiologically important enzyme dopamine [Beta]-monooxygenase and in several other secreted and transmembrane proteins such as SDR2 and CG-6. This domain was predicted to adopt an all-[Beta] fold with seven or eight strands and might function as a module mediating a range of extracellular adhesive interactions.

Sequence profile analysis was used to detect a conserved globular domain in several proteins including deltex, Trip 12 and poly-ADP-ribose polymerase homologs. It was named the WWE domain after its most conserved residues and is predicted to mediate specific protein-protein interactions in ubiquitin and ADP-ribose conjugation systems.

In a search for regulatory genes affecting the targeting of the condensin complex to chromatin in Saccharomyces cerevisiae, we identified a member of the adenovirus protease family, SMT4. SMT4 over-expression suppresses the temperature-sensitive conditional lethal phenotype of smc2-6, but not smc2-8 or smc4-1. A disruption allele of SMT4 has a prominent chromosome phenotype: impaired targeting of Smc4p-GFP to rDNA chromatin. Site-specific mutagenesis of the predicted protease active site cysteine and histidine residues of Smt4p abolishes the SMT4 function in vivo. The previously uncharacterized SIZ1 (SAP and Miz) gene, which encodes a protein containing a predicted DNA-binding SAP module and a Miz finger, is identified as a bypass suppressor of the growth defect associated with the SMT4 disruption. The SIZ1 gene disruption is synthetically lethal with the SIZ2 deletion. We propose that SMT4, SIZ1, and SIZ2 are involved in a novel pathway of chromosome maintenance.

Sequence profile searches were used to identify an ancient domain in Thil-like thiouridine synthases, conserved RNA methylases, archaeal pseudouridine synthases and several uncharacterized proteins. We predict that this domain is an RNA-binding domain that adopts an [Alpha]/[Beta] fold similar to that found in the C-terminal domain of translation initiation factor 3 and ribosomal protein S8.

The chromophore-binding domains of phytochromes and the cGMP-binding domains of cGMP-specific phosphodiesterases (PDEs) are classified as members of a large group of homologous domains. GAF and PAS domains including the chromophore binding proactive yellow protein and heme-binding fixL possess similar characteristics. Each domain type has regulatory roles in light and redox transduction pathways which includes chromophore-binding members.

A novel, 200-230 amino acid extracellular domain was identified in the plant cytokinin receptor Cre1, in the receptor-histidine kinase DhkA and the adenylyl cyclase Acg from the slime mold Dictyostelium discoideum, and in a variety of other receptor-like proteins from bacteria and eukaryotes. The domain is predicted to bind diverse low molecular weight ligands, such as the cytokinin-like adenine derivatives or peptides, and mediate signal transduction through the respective receptors.