The mechanistic and evolutionary aspects of the 2'- and 3'-OH paradigm in biosynthetic machinery.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: The translation machinery underlies a multitude of biological processes within the cell. The design and implementation of the modern translation apparatus on even the simplest course of action is extremely complex, and involves different RNA and protein factors. According to the "RNA world" idea, the critical link in the translation machinery may be assigned to an adaptor tRNA molecule. Its exceptional functional and structural characteristics are of primary importance in understanding the evolutionary relationships among all these macromolecular components. PRESENTATION OF THE HYPOTHESIS: The 2'-3' hydroxyls of the tRNA A76 constitute chemical groups of critical functional importance, as they are implicated in almost all phases of protein biosynthesis. They contribute to: a) each step of the tRNA aminoacylation reaction catalyzed by aminoacyl-tRNA synthetases (aaRSs); b) the isomerase activity of EF-Tu, involving a mixture of the 2'(3')- aminoacyl tRNA isomers as substrates, thereby producing the required combination of amino acid and tRNA; and c) peptide bond formation at the peptidyl transferase center (PTC) of the ribosome. We hypothesize that specific functions assigned to the 2'-3' hydroxyls during peptide bond formation co-evolved, together with two modes of attack on the aminoacyl-adenylate carbonyl typical for two classes of aaRSs, and alongside the isomerase activity of EF-Tu. Protein components of the translational apparatus are universally recognized as being of ancient origin, possibly replacing RNA-based enzymes that may have existed before the last universal common ancestor (LUCA). We believe that a remnant of these processes is still imprinted on the organization of modern-day translation. TESTING AND IMPLICATIONS OF THE HYPOTHESIS: Earlier publications indicate that it is possible to select ribozymes capable of attaching the aa-AMP moiety to RNA molecules. The scenario described herein would gain general acceptance, if a ribozyme able to activate the amino acid and transfer it onto the terminal ribose of the tRNA, would be found in any life form, or generated in vitro. Interestingly, recent studies have demonstrated the plausibility of using metals, likely abandoned under primordial conditions, as biomimetic catalysts of the aminoacylation reaction

MetaGeneAnnotator: Detecting Species-Specific Patterns of Ribosomal Binding Site for Precise Gene Prediction in Anonymous Prokaryotic and Phage Genomes

Recent advances in DNA sequencers are accelerating genome sequencing, especially in microbes, and complete and draft genomes from various species have been sequenced in rapid succession. Here, we present a comprehensive gene prediction tool, the MetaGeneAnnotator (MGA), which precisely predicts all kinds of prokaryotic genes from a single or a set of anonymous genomic sequences having a variety of lengths. The MGA integrates statistical models of prophage genes, in addition to those of bacterial and archaeal genes, and also uses a self-training model from input sequences for predictions. As a result, the MGA sensitively detects not only typical genes but also atypical genes, such as horizontally transferred and prophage genes in a prokaryotic genome. In this paper, we also propose a novel approach for analyzing the ribosomal binding site (RBS), which enables us to detect species-specific patterns of the RBSs. The MGA has the ingenious RBS model based on this approach, and precisely predicts translation starts of genes. The MGA also succeeds in improving prediction accuracies for short sequences by using the adapted RBS models (96% sensitivity and 93% specificity for 700 bp fragments). These features of the MGA expedite wide ranges of microbial genome studies, such as genome annotations and metagenome analyses

Common Peptides Study of Aminoacyl-tRNA Synthetases

Aminoacyl tRNA synthetases (aaRSs) constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains.We utilized the Common Peptides (CPs) framework, based on extracted deterministic motifs from all aaRSs, to study family-specific properties. We identified novel aaRS–class related signatures that may supplement the current classification methods and provide a basis for identifying functional regions specific to each aaRS class. We exploited the space spanned by the CPs in order to identify similarities between aaRS families that are not observed using sequence alignment methods, identifying different inter-aaRS associations across different kingdom of life. We explored the evolutionary history of the aaRS families and evolutionary origins of the mitochondrial aaRSs. Lastly, we showed that prevalent CPs significantly overlap known catalytic and binding sites, suggesting that they have meaningful functional roles, as well as identifying a motif shared between aaRSs and a the Biotin-[acetyl-CoA carboxylase] synthetase (birA) enzyme overlapping binding sites in both families.The study presents the multitude of ways to exploit the CP framework in order to extract meaningful patterns from the aaRS super-family. Specific CPs, discovered in this study, may play important roles in the functionality of these enzymes. We explored the evolutionary patterns in each aaRS family and tracked remote evolutionary links between these families

Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14

The modified nucleosides N2-methylguanosine and N22-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m2G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m2G at position 6 in tRNACys. The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m2G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20–30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m2G6 tRNA modification

An ancient family of SelB elongation factor-like proteins with a broad but disjunct distribution across archaea

<p>Abstract</p> <p>Background</p> <p>SelB is the dedicated elongation factor for delivery of selenocysteinyl-tRNA to the ribosome. In archaea, only a subset of methanogens utilizes selenocysteine and encodes archaeal SelB (aSelB). A SelB-like (aSelBL) homolog has previously been identified in an archaeon that does not encode selenosysteine, and has been proposed to be a pyrrolysyl-tRNA-specific elongation factor (EF-Pyl). However, elongation factor EF-Tu is capable of binding archaeal Pyl-tRNA in bacteria, suggesting the archaeal ortholog EF1A may also be capable of delivering Pyl-tRNA to the ribosome without the need of a specialized factor.</p> <p>Results</p> <p>We have phylogenetically characterized the aSelB and aSelBL families in archaea. We find the distribution of aSelBL to be wider than both selenocysteine and pyrrolysine usage. The aSelBLs also lack the carboxy terminal domain usually involved in recognition of the selenocysteine insertion sequence in the target mRNA. While most aSelBL-encoding archaea are methanogenic Euryarchaea, we also find aSelBL representatives in Sulfolobales and Thermoproteales of Crenarchaea, and in the recently identified phylum Thaumarchaea, suggesting that aSelBL evolution has involved horizontal gene transfer and/or parallel loss. Severe disruption of the GTPase domain suggests that some family members may employ a hitherto unknown mechanism of nucleotide hydrolysis, or have lost their GTPase ability altogether. However, patterns of sequence conservation indicate that aSelBL is still capable of binding the ribosome and aminoacyl-tRNA.</p> <p>Conclusions</p> <p>Although it is closely related to SelB, aSelBL appears unlikely to either bind selenocysteinyl-tRNA or function as a classical GTP hydrolyzing elongation factor. We propose that following duplication of aSelB, the resultant aSelBL was recruited for binding another aminoacyl-tRNA. In bacteria, aminoacylation with selenocysteine is essential for efficient thermodynamic coupling of SelB binding to tRNA and GTP. Therefore, change in tRNA specificity of aSelBL could have disrupted its GTPase cycle, leading to relaxation of selective pressure on the GTPase domain and explaining its apparent degradation. While the specific role of aSelBL is yet to be experimentally tested, its broad phylogenetic distribution, surpassing that of aSelB, indicates its importance.</p

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Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability. DOI: http://dx.doi.org/10.7554/eLife.02501.00

The Rodin-Ohno hypothesis that two enzyme superfamilies descended from one ancestral gene: an unlikely scenario for the origins of translation that will not be dismissed

Background Because amino acid activation is rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in understanding the origin of the genetic code. Two unrelated classes (I and II) of contemporary aminoacyl-tRNA synthetases (aaRS) now translate the code. Observing that codons for the most highly conserved, Class I catalytic peptides, when read in the reverse direction, are very nearly anticodons for Class II defining catalytic peptides, Rodin and Ohno proposed that the two superfamilies descended from opposite strands of the same ancestral gene. This unusual hypothesis languished for a decade, perhaps because it appeared to be unfalsifiable. Results The proposed sense/antisense alignment makes important predictions. Fragments that align in antiparallel orientations, and contain the respective active sites, should catalyze the same two reactions catalyzed by contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called Urzymes after Ur = primitive, authentic, plus enzyme and representing ~20% of the contemporary structures, can be expressed and exhibit high, proportionate rate accelerations for both amino-acid activation and tRNA acylation. A major fraction (60%) of the catalytic rate acceleration by contemporary synthetases resides in segments that align sense/antisense. Bioinformatic evidence for sense/antisense ancestry extends to codons specifying the invariant secondary and tertiary structures outside the active sites of the two synthetase classes. Peptides from a designed, 46-residue gene constrained by Rosetta to encode Class I and II ATP binding sites with fully complementary sequences both accelerate amino acid activation by ATP ~400 fold. Conclusions Biochemical and bioinformatic results substantially enhance the posterior probability that ancestors of the two synthetase classes arose from opposite strands of the same ancestral gene. The remarkable acceleration by short peptides of the rate-limiting step in uncatalyzed protein synthesis, together with the synergy of synthetase Urzymes and their cognate tRNAs, introduce a new paradigm for the origin of protein catalysts, emphasize the potential relevance of an operational RNA code embedded in the tRNA acceptor stems, and challenge the RNA-World hypothesis. Reviewers This article was reviewed by Dr. Paul Schimmel (nominated by Laura Landweber), Dr. Eugene Koonin and Professor David Ardell