The non-monophyletic origin of the tRNA molecule and the origin of genes only after the evolutionary stage of the last universal common ancestor (LUCA)

A model has been proposed suggesting that the tRNA molecule must have originated by direct duplication of an RNA hairpin structure [Di Giulio, M., 1992. On the origin of the transfer RNA molecule. J. Theor. Biol. 159, 199-214]. A non-monophyletic origin of this molecule has also been theorized [Di Giulio, M., 1999. The non-monophyletic origin of tRNA molecule. J. Theor. Biol. 197, 403-414]. In other words, the tRNA genes evolved only after the evolutionary stage of the last universal common ancestor (LUCA) through the assembly of two minigenes codifying for different RNA hairpin structures, which is what the exon theory of genes suggests when it is applied to the model of tRNA origin. Recent observations strongly corroborate this theorization because it has been found that some tRNA genes are completely separate in two minigenes codifying for the 5' and 3' halves of this molecule [Randau, L., et al., 2005a. Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5'- and 3'-halves. Nature 433, 537-541]. In this paper it is shown that these tRNA genes codifying for the 5' and 3' halves of this molecule are the ancestral form from which the tRNA genes continuously codifying for the complete tRNA molecule are thought to have evolved. This, together with the very existence of completely separate tRNA genes codifying for their 5' and 3' halves, proves a non-monophyletic origin for tRNA genes, as a monophyletic origin would exclude the existence of these genes which have, on the contrary, been observed. Here the polyphyletic origin of genes codifying for proteins is also suggested and discussed. Moreover, a hypothesis is advanced to suggest that the LUCA might have had a fragmented genome made up of RNA and the possibility that 'Paleokaryotes' may exist is outlined. Finally, the characteristic of the indivisibility of homology that these polyphyletic origins seem to remove at the sequence level is discussed.     

The Presence in tRNA Molecule Sequences of the Double Hairpin,an Evolutionary Stage Through Which the Origin of this Molecule is Thought to have Passed

We analyse 6,810 tRNAs, calculating the free energy of the corresponding double hairpin and ‘cigar’ secondary structures, for which we find a high thermodynamic and statistical significance. We also analyse these tRNAs for similarity and complementarity of their 5′ and 3′ halves or segments of them in intra-and inter-molecular comparisons. We find very clear signs that the two halves of tRNAs had an evident evolutionary relationship, although it is not totally clear whether this was a relationship of homology or complementarity between the 5′ and 3′ halves of tRNAs, even if there is strong evidence in favour of the homology hypothesis. Overall, these data favour models for the origin of the tRNA molecule postulating that a duplication event involving a hairpin structure as a precursor was involved in the origin of this molecule. Moreover, we interpret these results and favour the hypothesis that sees the assembly of two hairpin structures sharing a homology relationship as the intermediate evolutionary stage preceding the appearance of the cloverleaf structure of tRNA.     

tRNA Creation by Hairpin Duplication

Many studies have suggested that the modern cloverleaf structure of tRNA may have arisen through duplication of a primordial hairpin, but the timing of this duplication event has been unclear. Here we measure the level of sequence identity between the two halves of each of a large sample of tRNAs and compare this level to that of chimeric tRNAs constructed either within or between groups defined by phylogeny and/or specificity. We find that actual tRNAs have significantly more matches between the two halves than do random sequences that can form the tRNA structure, but there is no difference in the average level of matching between the two halves of an individual tRNA and the average level of matching between the two halves of the chimeric tRNAs in any of the sets we constructed. These results support the hypothesis that the modern tRNA cloverleaf arose from a single hairpin duplication prior to the divergence of modern tRNA specificities and the three domains of life. [Reviewing Editor: Dr. Niles Lehman]     

The Origin of the 5S Ribosomal RNA Molecule Could Have Been Caused by a Single Inverse Duplication: Strong Evidence from Its Sequences

The secondary structure of the 5S ribosomal RNA (5S rRNA) molecule shows a high degree of symmetry. In order to explain the origin of this symmetry, it has been conjectured that one half of the 5S rRNA molecule was its precursor and that an indirect duplication of this precursor created the other half and thus the current symmetry of the molecule. Here, we have subjected to an empirical test both the indirect duplication model, analysing a total of 684 5S rRNA sequences for complementarity between the two halves of the 5S rRNA, and the direct duplication model analysing in this case the similarity between the two halves of this molecule. In intra- and inter-molecule and intra- and inter-domain comparisons, we find a high statistical support to the hypothesis of a complementarity relationship between the two halves of the 5S rRNA molecule, denying vice versa the hypothesis of similarity between these halves. Therefore, these observations corroborate the indirect duplication model at the expense of the direct duplication model, as reason of the origin of the 5S rRNA molecule. More generally, we discuss and favour the hypothesis that all RNAs and proteins, which present symmetry, did so through gene duplication and not by gradualistic accumulation of few monomers or segments of molecule into a gradualistic growth process. This would be the consequence of the very high propensity that nucleic acids have to be subjected to duplications.     

The origin of the tRNA molecule: implications for the origin of protein synthesis

A model for tRNA molecule origin is discussed. The model postulates that this molecule originated simply by direct duplication (and subsequent evolution) of a gene coding for an RNA hairpin structure, which can thus be hypothesized as the evolutionary precursor of the tRNA molecule. The main properties are defined for these hairpin structures and it is suggested that these structures might have housed, near their 3' end, anticodons that were transferred to the loop of the tRNA anticodon during duplication of the hairpin structures. Moreover, the main characteristics are given for the evolutionary intermediary formed by direct duplication of the hairpin structure, i.e. the double hairpin. The evolutionary stages envisaged by this model for tRNA origin seem to naturally imply some evolutionary transitions through which the origin of protein synthesis passed. Finally, some strong historical evidence is provided to corroborate the model.     

The non-monophyletic origin of the tRNA molecule

The hypothesis that the tRNA molecule may have originated from the assembly of two similar RNA hairpin structures is utilised to understand the evolutionary period in which this molecule originated. Consistent with the exon theory of genes is the observation that the introns in tRNA genes are found almost exclusively in the anticodon loop and "stitched together" the two halves of the molecule, which originally may have been simply two hairpin structures and which can still be observed in the three-dimensional structure of tRNAs. This theory therefore considers these hairpin structures as minigenes on which complex protein synthesis may have been achieved. This in turn leads to the belief that the organisation of the genetic code may have been determined by use of the hairpin structures but not the complete tRNA molecule. In view of this, it can be conjectured that tRNA molecules might have been assembled only after the establishment of the main phyletic lines. If this is all true, then the origin of the tRNA molecule might have been non-monophyletic, i.e. a tRNA specific for a certain amino acid might have been assembled in different phyletic lines with a second and different hairpin structure. This leads to the belief that tRNAs specific for different amino acids but belonging to the same phyletic line might have been more similar to one another than to tRNAs specific for the same amino acid but belonging to different phyletic lines. This prediction seems to be supported by phylogenetic analysis making major use of the bootstrap technique performed on the tRNA sequences and by analysis already existing in the literature which supports the non-monophyletic origin of the tRNA molecule. The main conclusion of this paper is that if the tRNA molecule was assembled in the main phyletic lines this would imply a still rapidly evolving translation apparatus which, in turn, seems to imply that the last universal common ancestor was a progenote.     

Permuted tRNA genes of Cyanidioschyzon merolae, the origin of the tRNA molecule and the root of the Eukarya domain

An evolutionary analysis is conducted on the permuted tRNA genes of Cyanidioschyzon merolae, in which the 5′ half of the tRNA molecule is codified at the 3′ end of the gene and its 3′ half is codified at the 5′ end. This analysis has shown that permuted genes cannot be considered as derived traits but seem to possess characteristics that suggest they are ancestral traits, i.e. they originated when tRNA molecule genes originated for the first time. In particular, if the hypothesis that permuted genes are a derived trait were true, then we should not have been able to observe that the most frequent class of permuted genes is that of the anticodon loop type, for the simple reason that this class would derive by random permutation from a class of non-permuted tRNA genes, which instead is the rarest. This would not explain the high frequency with which permuted tRNA genes with perfectly separate 5′ and 3′ halves were observed. Clearly the mechanism that produced this class of permuted genes would envisage the existence, in an advanced stage of evolution, of minigenes codifying for the 5′ and 3′ halves of tRNAs which were assembled in a permuted way at the origin of the tRNA molecule, thus producing a high frequency of permuted genes of the class here referred. Therefore, this evidence supports the hypothesis that the genes of the tRNA molecule were assembled by minigenes codifying for hairpin-like RNA molecules, as suggested by one model for the origin of tRNA [Di Giulio, M., 1992. On the origin of the transfer RNA molecule. J. Theor. Biol. 159, 199–214; Di Giulio, M., 1999. The non-monophyletic origin of tRNA molecule. J. Theor. Biol. 197, 403–414]. Moreover, the late assembly of the permuted genes of C. merolae, as well as their ancestrality, strengthens the hypothesis of the polyphyletic origins of these genes. Finally, on the basis of the uniqueness and the ancestrality of these permuted genes, I suggest that the root of the Eukarya domain is in the super-ensemble of the Plantae and that the Rhodophyta to which C. merolae belongs are the first line of divergence.     

Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins

The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.     

Formal Proof that the Split Genes of tRNAs of <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis> Are an Ancestral Character

A proof is given that the genes of the tRNA molecule of Nanoarchaeum equitans split into the 5′ and 3′ halves are an ancestral trait. First, the existence of a natural succession of evolutionary stages will be proven, formed in the order of the three gene structures of tRNAs known today: (i) the split genes of tRNAs, (ii) the genes of tRNAs with introns, and (iii) the genes of tRNAs continuously codifying for the tRNA molecule. This succession of evolutionary stages identifies the split genes of tRNAs as a pleisiomorphic character. The proof that this succession of evolutionary stages is, moreover, true is performed by proving that all the possible remaining five successions of evolutionary stages are false. Indeed, the succession of evolutionary stages considering split genes as a derived character turns out to be false in that the increase in complexity inherent to this succession cannot be justified by the split genes of tRNAs because these could not have conferred any selective advantage justifying this increase in complexity. Furthermore, genetic drift is unable to explain the evolution of split genes of tRNAs because of the enormous genetic effective size of the population observed in these organisms. The remaining four successions of evolutionary stages are also false because: (i) they are not natural successions of evolutionary stages, (ii) the absolute observed frequencies of these evolutionary stages are such as to exclude categorically that they might be natural successions of evolutionary stages, and also (iii) two of these are falsified by the fact that they do not place the evolutionary stage of genes of tRNAs with introns in a close evolutionary relationship with that of the split genes of tRNAs which can, instead, be proven to have a close evolutionary link. Therefore, there remains only the succession of evolutionary stages considering the split genes of tRNAs codifying for the 5′ and 3′ halves, as a pleisiomorphic character, as the only succession compatible with all the arguments presented in this article and as the one that actually operated during the evolution of the tRNA molecule. This proof has two very important implications. One regards how the tRNA molecule originated; considering how tRNA originated as the union of two hairpin-like structures, the split genes of tRNAs might be the transition stage through which the evolution of this molecule passed. The other regards when the genes of tRNAs originated, reaching the conclusion that the origin of these genes is polyphyletic, i.e. not monophyletic and hence contrary to the assumptions of the current paradigm.     

Ligation of endogenous tRNA 3' half molecules to their corresponding 5' halves via 2'-phosphomonoester,3',5'-phosphodiester bonds in extracts of Chlamydomonas

tRNA preparations from Chlamydomonas and wheat germ contain small amounts of tRNA 5' halves and corresponding 3' halves. Incubation of cell-free extracts from the two sources with [γ-³²P]ATP yielded 5'-³²P-labeled tRNA 3' halves which were joined to their corresponding 5' counterparts to form mature tRNA containing 2'-phosphomonoester,3', 5'-phosphodiester bonds. tRNA 3' halves labelled with T4 kinase were purified, sequenced and also joined to their 5' counterparts. It is proposed that these tRNA halves may be intermediates of the tRNA splicing process, and that the RNA kinase and ligase activities observed here are part of the tRNA splicing complex.     

Sequence evidence in the archaeal genomes that tRNAs emerged through the combination of ancestral genes as 5' and 3' tRNA halves

The discovery of separate 5' and 3' halves of transfer RNA (tRNA) molecules-so-called split tRNA-in the archaeal parasite Nanoarchaeum equitans made us wonder whether ancestral tRNA was encoded on 1 or 2 genes. We performed a comprehensive phylogenetic analysis of tRNAs in 45 archaeal species to explore the relationship between the three types of tRNAs (nonintronic, intronic and split). We classified 1953 mature tRNA sequences into 22 clusters. All split tRNAs have shown phylogenetic relationships with other tRNAs possessing the same anticodon. We also mimicked split tRNA by artificially separating the tRNA sequences of 7 primitive archaeal species at the anticodon and analyzed the sequence similarity and diversity of the 5' and 3' tRNA halves. Network analysis revealed specific characteristics of and topological differences between the 5' and 3' tRNA halves: the 5' half sequences were categorized into 6 distinct groups with a sequence similarity of >80%, while the 3' half sequences were categorized into 9 groups with a higher sequence similarity of >88%, suggesting different evolutionary backgrounds of the 2 halves. Furthermore, the combinations of 5' and 3' halves corresponded with the variation of amino acids in the codon table. We found not only universally conserved combinations of 5'-3' tRNA halves in tRNA(iMet), tRNA(Thr), tRNA(Ile), tRNA(Gly), tRNA(Gln), tRNA(Glu), tRNA(Asp), tRNA(Lys), tRNA(Arg) and tRNA(Leu) but also phylum-specific combinations in tRNA(Pro), tRNA(Ala), and tRNA(Trp). Our results support the idea that tRNA emerged through the combination of separate genes and explain the sequence diversity that arose during archaeal tRNA evolution.     

Expression and characterization of the N-terminal half of antistasin, an anticoagulant protein derived from the leech Haementeria officinalis

Antistasin, a 15-kDa anticoagulant protein isolated from the salivary glands of the Mexican leech Haementeria officinalis, has been shown to be a potent inhibitor of factor Xa in the blood coagulation cascade. Antistasin possesses a twofold internal homology between the N- and C-terminal halves of the molecule, suggesting a gene duplication event in the evolution of the antistasin gene. This structural feature also suggests that either or both halves of the protein may possess biological activity if expressed as separate domains. Because the N-terminal domain contains a factor Xa P1-reactive site, we chose to express this domain in an insect cell baculovirus expression system. Characterization of this recombinant half antistasin molecule reveals that the N-terminal domain inhibits factor Xa in vitro, with a K(i) of 1.7 nM.     

Splicing of yeast tRNA precursors: structure of the reaction intermediates.

The intermediates of the yeast tRNA splicing reaction have been characterized. The intervening sequence is excised as an unique linear molecule. It has 5'-hydroxyl and 3'-phosphate termini. Correspondingly, the half-tRNA molecules are shown to have a 3'-phosphate terminus on the 5' half and 5'-hydroxyl terminus on the 3' half. These isolated halves have been shown to be active in the ligation step of tRNA splicing. Removal of the 3'-phosphate from the 5' half eliminates the ability of the 5' half to participate in ligation.     

血管生成素与tRNA片段化

tRNA主要功能是转运氨基酸参与蛋白质合成,在蛋白质生物合成过程中起着关键性的作用．近年来发现,tRNA是细胞内小RNA分子的重要来源,具有其它重要的生物学功能．来源于成熟tRNA分子的tRNA片段根据切割位置及生成机制的不同,主要分为两类：一类是tRNA半分子（tRNA halves）;另一类是较小的tRNA片段,称为tRFs( tRNA fragments)．在哺乳动物细胞中,tRNA半分子由血管生成素在tRNA分子反密码环处切割生成．本文主要针对tRNA半分子的加工机制、功能及在临床上的潜在应用进行综述．     

Assembly of a tRNA splicing complex: evidence for concerted excision and joining steps in splicing in vitro.

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.     

Several RNase T2 enzymes function in induced tRNA and rRNA turnover in the ciliate Tetrahymena

RNase T2 enzymes are produced by a wide range of organisms and have been implicated to function in diverse cellular processes, including stress-induced anticodon loop cleavage of mature tRNAs to generate tRNA halves. Here we describe a family of eight RNase T2 genes (RNT2A-RNT2H) in the ciliate Tetrahymena thermophila. We constructed strains lacking individual or combinations of these RNT2 genes that were viable but had distinct cellular and molecular phenotypes. In strains lacking only one Rnt2 protein or lacking a subfamily of three catalytically inactive Rnt2 proteins, starvation-induced tRNA fragments continued to accumulate, with only a minor change in fragment profile in one strain. We therefore generated strains lacking pairwise combinations of the top three candidates for Rnt2 tRNases. Each of these strains showed a distinct starvation-specific profile of tRNA and rRNA fragment accumulation. These results, the delineation of a broadened range of conditions that induce the accumulation of tRNA halves, and the demonstration of a predominantly ribonucleoprotein-free state of tRNA halves in cell extract suggest that ciliate tRNA halves are degradation intermediates in an autophagy pathway induced by growth arrest that functions to recycle idle protein synthesis machinery.     

Genomic organization of the human multidrug resistance (MDR1) gene and origin of P-glycoproteins

The MDR1 gene, responsible for multidrug resistance in human cells, encodes a broad specificity efflux pump (P-glycoprotein). P-glycoprotein consists of two similar halves, each half including a hydrophobic transmembrane region and a nucleotide-binding domain. On the basis of sequence homology between the N-terminal and C-terminal halves of P-glycoprotein, we have previously suggested that this gene arose by duplication of a primordial gene. We have now determined the complete intron/exon structure of the MDR1 gene by direct sequencing of cosmid clones and enzymatic amplification of genomic DNA segments. The MDR1 gene includes 28 introns, 26 of which interrupt the protein-coding sequence. Although both halves of the protein-coding sequence are composed of approximately the same number of exons, only two intron pairs, both within the nucleotide-binding domains, are located at conserved positions in the two halves of the protein. The other introns occur at different locations in the two halves of the protein and in most cases interrupt the coding sequence at different positions relative to the open reading frame. These results suggest that the P-glycoprotein arose by fusion of genes for two related but independently evolved proteins rather than by internal duplication.     

Gene duplications in the structural evolution of chymotrypsin.

Chymotrypsin and other members of the serine protease enzyme family have a structure built from two similar domains, each of which is a hydrogen-bonded barrel, containing six antiparallel strands of beta-sheet bonded in the order ABCFED-A …. The folding patterns of the domains have been re-examined by several newly improved shape comparison methods to see whether the barrels could have evolved by gene duplication, as proposed by Matthews and Blow (Birktoft &; Blow, 1972). The domains have a similar hydrogen-bond pattern, the same shear number (defined in this paper) for the twist of the barrel, and the cores of their β-sheets can be superimposed so that 46 topologically equivalent α-carbons fit within a root-mean-square distance of 2.43 Å and a larger set of 57 α-carbons fit within 3.4 Å. These results are highly significant when judged against shape comparisons of many other proteins with themselves, and give strong evidence for gene duplication. The duplication does not include any SS bridges.Both domains have a surprisingly symmetrical structure of two halves ABC, DEF paired round a dyad axis, and the half-domains are each made of two loops twisted in an L-shape, since the second strand (B or E) is bent into two halves B₁, B₂ or E₁, E₂. The cores of the four half-domains, each of 23 α-carbons, superimpose in pairs with root-mean-square distances ranging from 1.79 to 2.45 Å. In the entire molecule the half-domains are related by a screw dyad which converts domain I strands (ABC) (DEF) into domain II strands (DEF) (ABC) superimposing the six strands with a root-mean-square distance of 2.35 Å. These observations suggest that the Chymotrypsin barrel originally evolved from a closely-linked dimer of two intertwined half-domains which became united into one. domain by gene duplication. The enzyme evolved from a second dimer of two full domains and a second duplication. The bacterial protease B from Streptomyces griseus shows the same structural repeats and is consistent with the gene duplication hypothesis.Improved methods for shape comparison of proteins have been developed which are very fast and reliable.     

Translation of both complementary strands might govern early evolution of the genetic code

The updated structural and phylogenetic analyses of tRNA pairs with complementary anticodons provide independent support for our earlier finding, namely that these tRNA pairs concertedly show complementary second bases in the acceptor stem. Two implications immediately follow: first, that a tRNA molecule gained its present, complete, cloverleaf shape via duplication(s) of a shorter precursor. Second, that common ancestry is shared by two major components of the genetic code within the tRNA molecule--the classic code per se embodied in anticodon triplets, and the operational code of aminoacylation embodied primarily in the first three base pairs of the acceptor stems. In this communication we show that it might have been a double, sense-antisense, in-frame translation of the very first protein-encoding genes that directed the code's earliest expansion, thus preserving this fundamental dual-complementary link between acceptors and anticodons. Furthermore, the dual complementarity appears to be consistent with two mirror-symmetrical modes by which class I and II aminoacyl-tRNA synthetases recognize the cognate tRNAs--from the minor and major groove side of the acceptor stem, respectively.