An HflX-type GTPase from Sulfolobus solfataricus binds to the 50S ribosomal subunit in all nucleotide-bound states

HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint (1)H-(15)N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins.     

N2-methylation of guanosine at position 10 in tRNA is catalyzed by a THUMP domain-containing, S-adenosylmethionine-dependent methyltransferase, conserved in Archaea and Eukaryota

In sequenced genomes, genes belonging to the cluster of orthologous group COG1041 are exclusively, and almost ubiquitously, found in Eukaryota and Archaea but never in Bacteria. The corresponding gene products exhibit a characteristic Rossmann fold, S-adenosylmethionine-dependent methyltransferase domain in the C terminus and a predicted RNA-binding THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain in the N terminus. Recombinant PAB1283 protein from the archaeon Pyrococcus abyssi GE5, a member of COG1041, was purified and shown to behave as a monomeric 39-kDa entity. This protein (EC 2.1.1.32), now renamed (Pab)Trm-G10, which is extremely thermostable, forms a 1:1 complex with tRNA and catalyzes the adenosylmethionine-dependent methylation of the exocyclic amino group (N(2)) of guanosine located at position 10. Depending on the experimental conditions used, as well as the tRNA substrate tested, the enzymatic reaction leads to the formation of either N(2)-monomethyl (m(2)G) or N(2)-dimethylguanosine (m(2)(2)G). Interestingly, (Pab)Trm-G10 exhibits different domain organization and different catalytic site architecture from another, earlier characterized, tRNA-dimethyltransferase from Pyrococcus furiosus ((Pfu)Trm-G26, also known as (Pfu)Trm1, a member of COG1867) that catalyzes an identical two-step dimethylation of guanosine but at position 26 in tRNAs and is also conserved among all sequenced Eukaryota and Archaea. The co-occurrence of these two guanosine dimethyltransferases in both Archaea and Eukaryota but not in Bacteria is a hallmark of distinct tRNAs maturation strategies between these domains of life.     

Ribosomal protein-sequence block structure suggests complex prokaryotic evolution with implications for the origin of eukaryotes

Amino acid sequence alignments of orthologous ribosomal proteins found in Bacteria, Archaea, and Eukaryota display, relative to one another, an unusual segment or block structure, with major evolutionary implications. Within each of the prokaryotic phylodomains the sequences exhibit substantial similarity, but cross-domain alignments break up into (a) universal blocks (conserved in both phylodomains), (b) bacterial blocks (unalignable with any archaeal counterparts), and (c) archaeal blocks (unalignable with any bacterial counterparts). Sequences of those eukaryotic cytoplasmic riboproteins that have orthologs in both Bacteria and Archaea, exclusively match the archaeal block structure. The distinct blocks do not correlate consistently with any identifiable functional or structural feature including RNA and protein contacts. This phylodomain-specific block pattern also exists in a number of other proteins associated with protein synthesis, but not among enzymes of intermediary metabolism. While the universal blocks imply that modern Bacteria and Archaea (as defined by their translational machinery) clearly have had a common ancestor, the phylodomain-specific blocks imply that these two groups derive from single, phylodomain-specific types that came into existence at some point long after that common ancestor. The simplest explanation for this pattern would be a major evolutionary bottleneck, or other scenario that drastically limited the progenitors of modern prokaryotic diversity at a time considerably after the evolution of a fully functional translation apparatus. The vast range of habitats and metabolisms that prokaryotes occupy today would thus reflect divergent evolution after such a restricting event. Interestingly, phylogenetic analysis places the origin of eukaryotes at about the same time and shows a closer relationship of the eukaryotic ribosome-associated proteins to crenarchaeal rather than euryarchaeal counterparts.     

Disordered nucleiome: Abundance of intrinsic disorder in the DNA‐ and RNA‐binding proteins in 1121 species from Eukaryota,Bacteria and Archaea

Intrinsically disordered proteins (IDPs) are abundant in various proteomes, where they play numerous important roles and complement biological activities of ordered proteins. Among functions assigned to IDPs are interactions with nucleic acids. However, often, such assignments are made based on the guilty‐by‐association principle. The validity of the extension of these correlations to all nucleic acid binding proteins has never been analyzed on a large scale across all domains of life. To fill this gap, we perform a comprehensive computational analysis of the abundance of intrinsic disorder and intrinsically disordered domains in nucleiomes (～548 000 nucleic acid binding proteins) of 1121 species from Archaea, Bacteria and Eukaryota. Nucleiome is a whole complement of proteins involved in interactions with nucleic acids. We show that relative to other proteins in the corresponding proteomes, the DNA‐binding proteins have significantly increased disorder content and are significantly enriched in disordered domains in Eukaryotes but not in Archaea and Bacteria. The RNA‐binding proteins are significantly enriched in the disordered domains in Bacteria, Archaea and Eukaryota, while the overall abundance of disorder in these proteins is significantly increased in Bacteria, Archaea, animals and fungi. The high abundance of disorder in nucleiomes supports the notion that the nucleic acid binding proteins often require intrinsic disorder for their functions and regulation.     

COMBINED EFFECTS OF MACRONUTRIENTS AND METALS ON THE ADRIATIC GREEN ALGA ULVA RIGIDA C.AG.

Over 400 nuclear SSU rRNA sequences representing all orders of the Rhodophyta were aligned and analyzed using comparative sequence analysis. Numerous nucleotide positions and structural elements were found that delineated various taxonomic groups. The 1245 region ( E. coli numbering) contained a loop that differed in size between two conserved helices and clearly separated the Florideophyceae [3 nt (>95% of 268 sequences)], Bangiales [13 to 14 nt (100% of 116 sequences)] and remaining Bangiophyceae including the Cryptophyta nucleomorphs [four to eight nt (100% of 32 sequences)]. In addition, members of the Thoreaceae were found to have additional helices in the 650 and 1139 region of which a corresponding structure was not present in any other red algal SSU rRNA gene sequence. Base-pair and nucleotide signatures differentiated the Bangiales, Florideophyceae, Bangiophyceae (not including Bangiales) and Hildenbrandiales at three levels of comparison: within the Rhodophyta (>400 sequences), the Eukaryota (not including Rhodophyta;> 1300 sequences) and three kingdom (Archaea, Bacteria, 2 organelles, Eukaryota;> 7000 sequences). For example, all members of the Hildenbrandiales have a change in the base-pair 512:539 that is a region of functional importance. Sequences from the Eukaryota, Archaea, Bacteria and two organelles have a C:G or a U:A in this position whereas the Hildenbrandiales have a C:A pair. This analysis raises the possibility of utilizing structural features of nuclear SSU rRNA and sequence signatures to support and delineate phylogenetic clades within the Rhodophyta.     

STRUCTURAL AND SEQUENCE SIGNATURES OF NUCLEAR SSU RRNA DEFINE TAXONOMIC LEVELS WITHIN THE RHODOPHYTA

Over 400 nuclear SSU rRNA sequences representing all orders of the Rhodophyta were aligned and analyzed using comparative sequence analysis. Numerous nucleotide positions and structural elements were found that delineated various taxonomic groups. The 1245 region (E. coli numbering) contained a loop that differed in size between two conserved helices and clearly separated the Florideophyceae [3 nt (>95% of 268 sequences)], Bangiales [13 to 14 nt (100% of 116 sequences)] and remaining Bangiophyceae including the Cryptophyta nucleomorphs [four to eight nt (100% of 32 sequences)]. In addition, members of the Thoreaceae were found to have additional helices in the 650 and 1139 region of which a corresponding structure was not present in any other red algal SSU rRNA gene sequence. Base‐pair and nucleotide signatures differentiated the Bangiales, Florideophyceae, Bangiophyceae (not including Bangiales) and Hildenbrandiales at three levels of comparison: within the Rhodophyta (>400 sequences), the Eukaryota (not including Rhodophyta;> 1300 sequences) and three kingdom (Archaea, Bacteria, 2 organelles, Eukaryota;> 7000 sequences). For example, all members of the Hildenbrandiales have a change in the base‐pair 512:539 that is a region of functional importance. Sequences from the Eukaryota, Archaea, Bacteria and two organelles have a C:G or a U:A in this position whereas the Hildenbrandiales have a C:A pair. This analysis raises the possibility of utilizing structural features of nuclear SSU rRNA and sequence signatures to support and delineate phylogenetic clades within the Rhodophyta.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.     

Evolution of alpha-amylases: architectural features and key residues in the stabilization of the (beta/alpha)(8) scaffold

We provide a comprehensive analysis of the current enzymes with alpha-amylase activity (AAMYs) that belong to family 13 glycoside hydrolase (GH-13; 144 Archaea, Bacteria, and Eukaryota sequences from 87 different species). This study aims to further knowledge of the evolutionary molecular relationships among the sequences of their A and B domains with special emphasis on the correlation between what is observed in the structures and protein evolution. Multialignments for the A domain distinguish two clusters for sequences from Archaea organisms, eight for sequences from Bacteria organisms, and three for sequences from Eukaryota organisms. The clusters for Bacteria do not follow any strict taxonomic pathway; in fact, they are rather scattered. When we compared the A domains of sequences belonging to different kingdoms, we found that various pairs of clusters were significantly similar. Using either sequence similarity with crystallized structures or secondary-structure prediction methods, we identified in all AAMYs the eight putative beta-strands that constitute the beta-sheet in the TIM barrel of the A domain and studied the packing in its interior. We also discovered a "hidden homology" in the TIM barrel, an invariant Gly located upstream in the sequence before the conserved Asp in beta-strand 3. This Gly precedes an alpha-helix and is actively involved in capping its N-terminal end with a capping box. In all cases, a Schellman motif caps the C-terminal end of this helix.     

Horizontal Gene Transfer in Aminoacyl-tRNA Synthetases Including Leucine-Specific Subtypes

Aminoacyl-tRNA synthetases catalyze a fundamental reaction for the flow of genetic information from RNA to protein. Their presence in all organisms known today highlights their important role in the early evolution of life. We investigated the evolutionary history of aminoacyl-tRNA synthetases on the basis of sequence data from more than 200 Archaea, Bacteria, and Eukaryota. Phylogenetic profiles are in agreement with previous observations that many genes for aminoacyl-tRNA synthetases were transferred horizontally between species from all domains of life. We extended these findings by a detailed analysis of the history of leucyl-tRNA synthetases. Thereby, we identified a previously undetected case of horizontal gene transfer from Bacteria to Archaea based on phylogenetic profiles, trees, and networks. This means that, finally, the last subfamily of aminoacyl-tRNA synthetases has lost its exceptional position as the sole subfamily that is devoid of horizontal gene transfer. Furthermore, the leucyl-tRNA synthetase phylogenetic tree suggests a dichotomy of the archaeal/eukaryotic-cytosolic and bacterial/eukaryotic-mitochondrial proteins. We argue that the traditional division of life into Prokaryota (non-chimeric) and Eukaryota (chimeric) is favorable compared to Woese’s trichotomy into Archaea/Bacteria/Eukaryota. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.     

Functional characterization of EngA(MS), a P-loop GTPase of Mycobacterium smegmatis

Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of different mycobacterial species indicates the presence of multiple P-loop GTPases that exhibit highly conserved motifs. However, an exact function of most of these GTPases in mycobacteria remains elusive. In the present study we characterized the function of a P-loop GTPase in mycobacteria by employing an EngA homologue from Mycobacterium smegmatis, encoded by an open reading frame, designated as MSMEG_3738. Amino acid sequence alignment and phylogenetic analysis suggest that MSMEG_3738 (termed as EngA(MS)) is highly conserved in mycobacteria. Homology modeling of EngA(MS) reveals a cloverleaf structure comprising of α/β fold typical to EngA family of GTPases. Recombinant EngA(MS) purified from E. coli exhibits a GTP hydrolysis activity which is inhibited by the presence of GDP. Interestingly, the EngA(MS) protein is co-eluted with 16S and 23S ribosomal RNA during purification and exhibits association with 30S, 50S and 70S ribosomal subunits. Further studies demonstrate that GTP is essential for interaction of EngA(MS) with 50S subunit of ribosome and specifically C-terminal domains of EngA(MS) are required to facilitate this interaction. Moreover, EngA(MS) devoid of N-terminal region interacts well with 50S even in the absence of GTP, indicating a regulatory role of the N-terminal domain in EngA(MS)-50S interaction.     

The first determination of pseudouridine residues in 23S ribosomal RNA from hyperthermophilic Archaea Sulfolobus acidocaldarius

We describe the first identification of pseudouridine (Psi) residues in ribosomal RNA (23S rRNA) of an hyperthermophilic Archaea Sulfolobus acidocaldarius. In contrast to Eucarya rRNA, only six Psi residues were detected, which is rather close to the situation in Bacteria. However, three modified positions (Psi(2479), Psi(2535) and Psi(2550)) are unique for S. acidocaldarius. Two Psi residues at positions 2060 and 2594 are universally conserved, while one other Psi (position 2066) is also common to Eucarya. Taken together the results argue against the conservation of Psi-synthases between Archaea and Bacteria and provide a basis for the search of snoRNA-like guides for Psi formation in Archaea.     

Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.     

The Evolutionary History of the Structure of 5S Ribosomal RNA

5S rRNA is the smallest nucleic acid component of the large ribosomal subunit, contributing to ribosomal assembly, stability, and function. Despite being a model for the study of RNA structure and RNA–protein interactions, the evolution of this universally conserved molecule remains unclear. Here, we explore the history of the three-domain structure of 5S rRNA using phylogenetic trees that are reconstructed directly from molecular structure. A total of 46 structural characters describing the geometry of 666 5S rRNAs were used to derive intrinsically rooted trees of molecules and molecular substructures. Trees of molecules revealed the tripartite nature of life. In these trees, superkingdom Archaea formed a paraphyletic basal group, while Bacteria and Eukarya were monophyletic and derived. Trees of molecular substructures supported an origin of the molecule in a segment that is homologous to helix I (α domain), its initial enhancement with helix III (β domain), and the early formation of the three-domain structure typical of modern 5S rRNA in Archaea. The delayed formation of the branched structure in Bacteria and Eukarya lends further support to the archaeal rooting of the tree of life. Remarkably, the evolution of molecular interactions between 5S rRNA and associated ribosomal proteins inferred from a census of domain structure in hundreds of genomes established a tight relationship between the age of 5S rRNA helices and the age of ribosomal proteins. Results suggest 5S rRNA originated relatively quickly but quite late in evolution, at a time when primordial metabolic enzymes and translation machinery were already in place. The molecule therefore represents a late evolutionary addition to the ribosomal ensemble that occurred prior to the early diversification of Archaea.     

On the Occurrence of Horizontal Gene Transfer Among an Arbitrarily Chosen Group of 26 Genes

The deduced amino acid sequences from 1200 Haemophilus influenzae genes was compared to a data set that contained the orfs from yeast, two different Archaea and the Gram+ and Gram− bacteria, Bacillus subtilis and Escherichia coli. The results of the comparison yielded a 26 orthologous gene set that had at least one representative from each of the four groups. A four taxa phylogenetic relationship for these 26 genes was determined. The statistical significance of each minimal tree was tested against the two alternative four taxa trees. The result was that four genes significantly supported the (Archaea, Eukaryota) (Gram+, Gram−) topology, two genes supported the one where Gram− and Eukaryota form a clade, and one gene supported the tree where Gram+ and Eukaryota define one clade. The remaining genes do not uniquely support any phylogeny, thereby collapsing the two central nodes into a single node. These are referred to as star phylogenies. I offer a new suggestion for the mechanism that gave rise to the star phylogenies. Namely, these are genes that are younger than the underlying lineages that currently harbor them. This hypothesis is examined with two proteins that display the star phylogeny; namely onithine transcarbamylase and tryptophan synthetase. It is shown, using the distance matrix rate test, that the rate of evolution of these two proteins is comparable to a control gene when rates are determined by comparing closely related species. This implies that the genes under comparison experience comparable functional constraint. However, when the genes from remotely related species are compared, a plateau is encountered. Since we see no unusual levels of functional constraint this plateau cannot be attributed to the divergence of the protein having reached saturation. The simplest explanation is that the genes displaying the star phylogenies were introduced after Archaea, Eukaryota, and Bacteria had diverged from one another. They presumably spread through life by horizontal gene transfer. Received: 12 July 2001 / Accepted: 27 July 2001     

Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases

Methyltransferase enzymes that use S-adenosylmethionine as a cofactor to catalyze 5-methyl uridine (m(5)U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, but are restricted to the Thermococcales and Nanoarchaeota groups amongst the Archaea. The RNA m(5)U methyltransferases appear to have arisen in Bacteria and were then dispersed by horizontal transfer of an rlmD-type gene to the Archaea and Eukaryota. The bacterium Escherichia coli has three gene paralogs and these encode the methyltransferases TrmA that targets m(5)U54 in tRNAs, RlmC (formerly RumB) that modifies m(5)U747 in 23S rRNA, and RlmD (formerly RumA) the archetypical enzyme that is specific for m(5)U1939 in 23S rRNA. The thermococcale archaeon Pyrococcus abyssi possesses two m(5)U methyltransferase paralogs, PAB0719 and PAB0760, with sequences most closely related to the bacterial RlmD. Surprisingly, however, neither of the two P. abyssi enzymes displays RlmD-like activity in vitro. PAB0719 acts in a TrmA-like manner to catalyze m(5)U54 methylation in P. abyssi tRNAs, and here we show that PAB0760 possesses RlmC-like activity and specifically methylates the nucleotide equivalent to U747 in P. abyssi 23S rRNA. The findings indicate that PAB0719 and PAB0760 originated as RlmD-type m(5)U methyltransferases and underwent changes in target specificity after their acquisition by a Thermococcales ancestor from a bacterial source.     

Site-2 protease regulated intramembrane proteolysis: sequence homologs suggest an ancient signaling cascade

Site-2 proteases (S2Ps) form a large family of membrane-embedded metalloproteases that participate in cellular signaling pathways through sequential cleavage of membrane-tethered substrates. Using sequence similarity searches, we extend the S2P family to include remote homologs that help define a conserved structural core consisting of three predicted transmembrane helices with traditional metalloprotease functional motifs and a previously unrecognized motif (GxxxN/S/G). S2P relatives were identified in genomes from Bacteria, Archaea, and Eukaryota including protists, plants, fungi, and animals. The diverse S2P homologs divide into several groups that differ in various inserted domains and transmembrane helices. Mammalian S2P proteases belong to the major ubiquitous group and contain a PDZ domain. Sequence and structural analysis of the PDZ domain support its mediating the sequential cleavage of membrane-tethered substrates. Finally, conserved genomic neighborhoods of S2P homologs allow functional predictions for PDZ-containing transmembrane proteases in extra-cytoplasmic stress response and lipid metabolism.