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 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.     

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.     

On the origin of the transfer RNA molecule.

Data and arguments are given in favour of the hypothesis that the primitive tRNA molecule may have originated from a direct duplication event involving one of the two halves of the tRNA molecule. It seems that a molecule capable of assuming a hairpin structure was involved as a precursor in this duplication. The two halves of the present tRNAs could, therefore, be considered as paralogous.     

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.     

The origin of the tRNA molecule: Independent data favor a specific model of its evolution

The properties, historical and empirical observations of a model of the origin of the tRNA molecule are discussed. This model would predict that this molecule originated by means of the assembly of two hairpin-like structures of RNA. The conclusion is that the model possesses a relevant part of the truth on the origin of the tRNA molecule.     

Molecular evolution of transfer RNA from two precursor hairpins: Implications for the origin of protein synthesis

In this paper we are going to present a model for the coevolution of major components of the protein synthesis machinery in a primordial RNA world. We propose that the essential prerequisites for RNA-based protein synthesis, i.e., tRNA-like molecules, ribozymic charging catalysts, small-subunit(SSU) rRNA, and large-subunit(LSU) rRNA, evolved from the same ancestral RNA molecule. Several arguments are considered which suggest that tRNA-like molecules were derived by tandem joining of template-flanking hairpin structures involved in replication control. It is further argued that the ancestors of contemporary group I tRNA introns catalyzed such hairpin joining reactions, themselves also giving rise to the ribosomal RNAs. Our model includes a general stereochemical principle for the interaction between ribozymes and hairpin-derived recognition structures, which can be applied to such seemingly different processes as RNA polymerization, aminoacylation, tRNA decoding, and peptidyl transfer, implicating a common origin for these fundamental functions. These and other considerations suggest that generation and evolution of tRNA were coupled to the evolution of synthetases, ribosomal RNAs, and introns from the beginning and have been a consequence arising from the original function of tRNA precursor hairpins as replication and recombination control elements. Correspondence to: T.P. Dick     

基因倍增和脊椎动物起源

有机体基因复制导致基因复杂性增加及其和脊椎动物起源的关系已经成为进化生物学研究的热点。20世纪70年代由Ohno提出后经Holland等修正的原始脊索动物经两轮基因组复制产生脊椎动物的假设目前已被广泛接受。脊椎动物起源和进化过程中发生过两轮基因组复制的主要证据有三点：（1）据估计脊椎动物基因组内编码基因数目大约相当于果蝇、海鞘等无脊椎动物的4倍;原口动物如果蝇和后口动物如头索动物文昌鱼的基因组大都只有单拷贝的基因,而脊椎动物的基因组则通常有4个同属于一个家族的基因。（2）无脊椎动物如节肢动物、海胆和头索动物文昌鱼都只有一个Hox基因簇,而脊椎动物除鱼类外,有7个具有Hox基因簇,其余都具有4个Hox基因簇。（3）基因作图证明,不但在鱼类和哺乳动物染色体广大片段上基因顺序相似,而且有证据显示哺乳动物基因组不同染色体之间存在相似性。据认为第一次基因倍增发生在脊椎动物与头索动物分开之后,第二次基因倍增发生在有颌类脊椎动物和无颌类脊椎动物分开以后。但是,基因是逐个发生倍增,还是通过基因组内某些DNA片段抑或整个基因组的加倍而实现的,目前还颇有争议。     

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.     

A Comparison Among the Models Proposed to Explain the Origin of the tRNA Molecule: A Synthesis

A comparison is made among all the models proposed to explain the origin of the tRNA molecule. The conclusion reached is that, for the model predicting that the tRNA molecule originated after the assembly of two hairpin-like structures, molecular fossils have been found in the half-genes of the tRNAs of Nanoarchaeum equitans. These might be the witnesses of the transition stage predicted by the model through which the evolution of the tRNA molecule passed, thus providing considerable corroboration for this model.     

A mechanistic view on the evolutionary origin for centrin-based control of centriole duplication

Mounting evidence implicates the protein centrin as a key regulator of centriole duplication, yet it remains to be determined just how centrin functions in this process. Recent studies suggest that centrin exerts both spatial and temporal control over centriole duplication through its role as a component of centriole precursor structures and through periodic cell-cycle specific changes in its abundance. Here, an overview of centrin and its role in centrosome dynamics is presented. Finally, a speculative model for just how centrin may operate to control centriole duplication is proposed with the intention to stimulate future advances in this area. This model provides an evolutionary basis for the preservation of essential features of the yeast spindle pole body (SPB) with the origin of the complex structure of the mammalian centriole.     

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.     

Ribosome evolution: Emergence of peptide synthesis machinery

Proteins, the main players in current biological systems, are produced on ribosomes by sequential amide bond (peptide bond) formations between amino-acid-bearing tRNAs. The ribosome is an exquisite super-complex of RNA-proteins, containing more than 50 proteins and at least 3 kinds of RNAs. The combination of a variety of side chains of amino acids (typically 20 kinds with some exceptions) confers proteins with extraordinary structure and functions. The origin of peptide bond formation and the ribosome is crucial to the understanding of life itself. In this article, a possible evolutionary pathway to peptide bond formation machinery (proto-ribosome) will be discussed, with a special focus on the RNA minihelix (primordial form of modern tRNA) as a starting molecule. Combining the present data with recent experimental data, we can infer that the peptidyl transferase center (PTC) evolved from a primitive system in the RNA world comprising tRNA-like molecules formed by duplication of minihelix-like small RNA.     

Evidence from glycine transfer RNA of a frozen accident at the dawn of the genetic code

Background

Transfer RNA (tRNA) is the means by which the cell translates DNA sequence into protein according to the rules of the genetic code. A credible proposition is that tRNA was formed from the duplication of an RNA hairpin half the length of the contemporary tRNA molecule, with the point at which the hairpins were joined marked by the canonical intron insertion position found today within tRNA genes. If these hairpins possessed a 3'-CCA terminus with different combinations of stem nucleotides (the ancestral operational RNA code), specific aminoacylation and perhaps participation in some form of noncoded protein synthesis might have occurred. However, the identity of the first tRNA and the initial steps in the origin of the genetic code remain elusive.

Results

Here we show evidence that glycine tRNA was the first tRNA, as revealed by a vestigial imprint in the anticodon loop sequences of contemporary descendents. This provides a plausible mechanism for the missing first step in the origin of the genetic code. In 448 of 466 glycine tRNA gene sequences from bacteria, archaea and eukaryote cytoplasm analyzed, CCA occurs immediately upstream of the canonical intron insertion position, suggesting the first anticodon (NCC for glycine) has been captured from the 3'-terminal CCA of one of the interacting hairpins as a result of an ancestral ligation.

Conclusion

That this imprint (including the second and third nucleotides of the glycine tRNA anticodon) has been retained through billions of years of evolution suggests Crick's 'frozen accident' hypothesis has validity for at least this very first step at the dawn of the genetic code.

Reviewers

This article was reviewed by Dr Eugene V. Koonin, Dr Rob Knight and Dr David H Ardell.     

Unique hairpin structures occurring at the replication origin of phage G4 DNA

Recently we reported that a DNA fragment, GCCAAAGC, forms an extraordinarily stable hairpin structure with two G-C pairs at the terminus and A-A-A stacked structure. The sequence is present at the replication origin of bacteriophage G4 ssDNA, and so on. Several kinds of possible hairpin structures, corresponding to the replication origin of phage G4, were synthesized and their secondary structures were examined. It was found that the fragments are able to form interconvertible hairpin structures depending on the length of the base-paired regions. The hairpin structure consisting of GCGAAAGC was not digested by the exonuclease activity of T4 DNA polymerase and it was stable enough to be only minimally bound by a single-stranded DNA binding protein.     

Human tyrosine tRNA is also internally cleavable by E. coli ribonuclease P RNA ribozyme in vitro

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA(Ala), tRNA(His), and tRNA(iMet)) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool detect destablized tRNA molecules from any species.     

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]     

Length Variation in Mitochondrial DNA of the Minnow Cyprinella Spiloptera

Length differences in animal mitochondrial DNA (mtDNA) are common, frequently due to variation in copy number of direct tandem duplications. While such duplications appear to form without great difficulty in some taxonomic groups, they appear to be relatively short-lived, as typical duplication products are geographically restricted within species and infrequently shared among species. To better understand such length variation, we have studied a tandem and direct duplication of approximately 260 bp in the control region of the cyprinid fish, Cyprinella spiloptera. Restriction site analysis of 38 individuals was used to characterize population structure and the distribution of variation in repeat copy number. This revealed two length variants, including individuals with two or three copies of the repeat, and little geographic structure among populations. No standard length (single copy) genomes were found and heteroplasmy, a common feature of length variation in other taxa, was absent. Nucleotide sequence of tandem duplications and flanking regions localized duplication junctions in the phenylalanine tRNA and near the origin of replication. The locations of these junctions and the stability of folded repeat copies support the hypothesized importance of secondary structures in models of duplication formation.     

Comparing two evolutionary mechanisms of modern tRNAs

All modern tRNA gene families have a high similarity in their primary structure, and share the same cloverleaf secondary structure and an inverted L tertiary structure, which provide the clues for the study of their origin and evolution. There are two important mechanisms of the tRNA sequences evolution. One is point mutation, another is complementary duplication method. Both of them are supported by some specific examples. To find out the superior one of the two mechanisms or find out the most suitable mechanism for modern tRNAs evolution, we constructed two types of networks, parallel and antiparallel networks, based on the two mechanisms respectively, and then compared the degree distribution and clustering coefficient of networks constructed by the tRNA sequences of the single anticodon group, single isoaccepting group, and the whole tRNAs group of the two types of networks. The result of the comparison seems consistent with the idea that modern tRNA sequences evolved primarily by the mechanism of complementary method, and point mutation is an important and indispensable auxiliary mechanism during the evolutionary event.