Evolution of tRNAs and tRNA genes in Acholeplasma laidlawii.

The genes for 22 tRNA species from Acholeplasma laidawii, belonging to the class Mollicutes (Mycoplasmas), have been cloned and sequenced. Sixteen genes are organized in 3 clusters consisting of eleven, three and two tRNA genes, respectively, and the other 6 genes exist as a single gene. The arrangement of tRNA genes in the 11-gene, the 3-gene and the 2-gene clusters reveals extensive similarity to several parts of the 21-tRNA or 16-tRNA gene cluster in Bacillus subtilis. The 11-gene cluster is also similar to the tRNA gene clusters found in other mycoplasma species, the 9-tRNA gene cluster in M.capricolum and in M.mycoides, and the 10-tRNA gene cluster in Spiroplasma meliferm. The results suggest that the tRNA genes in mycoplasmas have evolved from large tRNA gene clusters in the ancestral Gram-positive bacterial genome common to mycoplasmas and B.subtilis. The anticodon sequences including base modifications of 15 tRNA species from A.laidlawii were determined. The anticodon composition and codon-recognition patterns of A.laidlawii resemble those of Bacillus subtilis rather than those of other mycoplasma species.     

Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.     

Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria

The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.     

A cluster of nine tRNA genes between ribosomal gene operons in Bacillus subtilis.

A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rrnJ and rrnW in Bacillus subtilis has been cloned and sequenced. This cluster contains the genes for tRNA(UACVal), tRNA(UGUThr), tRNA(UUULys), tRNA(UAGLeu). tRNA(GCCGly), tRNA(UAALeu), tRNA(ACGArg), tRNA(UGGPro), and tRNA(UGCAla). The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnI and rrnH, and of the 5'-end of trnB, a cluster of 21 tRNA genes found immediately 3' to rrnB. Neither the tRNA(UAGLeu) gene nor its product has been found previously in B. subtilis. With the discovery of this new set of tRNA genes, a total of 60 such genes have now been found in B. subtilis. These known genes account for almost all of the tRNA hybridizing restriction fragments of the B. subtilis genome. The 60 known tRNA genes of B. subtilis code for only 28 different anticodons, compared with a total of 41 different anticodons for 78 tRNA genes in Escherichia coli. This may indicate that B. subtilis does not need as many anticodons because of more flexible translation rules, similar to the situation in Mycoplasma capricolum.     

Physical mapping of stable RNA genes in Bacillus subtilis using polymerase chain reaction amplification from a yeast artificial chromosome library.

A new approach for mapping genes which utilizes yeast artificial chromosome clones carrying parts of the Bacillus subtilis genome and the polymerase chain reaction technique is described. This approach was used to physically map stable RNA genes of B. subtilis. Results from over 400 polymerase chain reactions carried out with the yeast artificial chromosome clone library, using primers specific for the genes of interest and designed from published sequences, were collected. The locations of 10 known rRNA gene regions (rrnO, rrnA, rrnE, rrnD, rrnB, rrnJ-rrnW, and rrnI-rrnH-rrnG) have been determined by this method, and these results correlate with those observed by standard genetic mapping. All rRNA operons, except rrnB, are found between 0 and 90 degrees, while rrnB has been placed in the area of 270 degrees on the chromosome map. Also localized were the tRNA gene clusters associated with the following ribosomal operons: rrnB (21 tRNAs), rrnJ (9 tRNAs), rrnD (16 tRNAs), and rrnO and rrnA (2 internal tRNAs). A previously unmapped four-tRNA gene cluster, trnY, a tRNA gene region that is not associated with a ribosomal operon, was found near the origin of replication. The P-RNA gene, important for processing of tRNAs, was found between map locations 197 and 204 degrees.     

Codon reading scheme in Mycoplasma pneumoniae revealed by the analysis of the complete set of tRNA genes.

The 33 genes encoding the complete set of tRNA species in Mycoplasma pneumoniae have been cloned and sequenced. They are organized into 5 clusters in addition to 9 single genes. No redundant gene was found, indicating that 33 tRNAs correspond to 32 different anticodons and decode all 62 codons used in this organism. There is only one single tRNA for each of the Ala, Leu, Pro, and Val family boxes. Therefore, a simplified decoding system resembling that recently described for Mycoplasma capricolum (1) has to also exist in M.pneumoniae. However, analysis of the anticodon set and codon usage revealed features characteristic of the latter: (i) there is no obvious preference toward AT rich synonymous codons, (ii) CGG codons are assigned for arginine and are translated by tRNA Arg(UCG), and (iii) CNN or GNN anticodons are encountered in the Ser, Thr, Arg, and Gly family boxes. We thus propose that this codon-anticodon recognition pattern has emerged in the 'M.pneumoniae cluster' under a genomic economization strategy but without the influence of AT pressure.     

Highly repetitive tRNA(Pro)-tRNA(His) gene cluster from Photobacterium phosphoreum.

A DNA fragment comprising the four tRNA gene sequences of the Escherichia coli argT locus hybridized with two Sau3A-generated DNA fragments from the vibrio Photobacterium phosphoreum (ATCC 11040). Detailed sequence analysis of the longer fragment shows the following gene organization: 5'-promoter-tRNA(Pro)-tRNAPro-tRNA(Pro)-tRNA(His)-tRNA(Pro)-tRNA(Pro)- tRNA(His)-tRNA(Pro)-five pseudogenes derived from the upstream tRNAPro interspersed by putative Rho-independent terminators. This sequence demonstrates the presence of highly repetitive, tandem tRNA genes in a bacterial genome. Furthermore, a stretch of 304 nucleotides from this cluster was found virtually unchanged in the other (shorter) fragment which was previously sequenced. The two clusters together contain eight tRNA(Pro) pseudogenes and eight fully intact tRNA(Pro) genes, an unusually high number for a single eubacterial isoacceptor tRNA. These results show that the organization of some tRNA operons is highly variable in eubacteria.     

Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)- tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem-and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodialians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and- loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.      

Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes.

We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.     

Transfer RNA genes in the mitochondrial genome from a liverwort, Marchantia polymorpha: the absence of chloroplast-like tRNAs.

Twenty-nine genes for 27 species of tRNAs were deduced from the complete nucleotide sequence of the mitochondrial genome from a liverwort, Marchantia polymorpha. One to three species of tRNA genes corresponded to each of 20 amino acids including three species for leucine and arginine, two species for serine and glycine, and one for the rest of the amino acids. Interestingly, all tRNA genes were located in the semicircle of the liverwort mitochondrial genome except for the trnY and trnR genes. The region containing these tRNA genes was originally duplicated, and two trnR genes have diverged from each other. On the other hand, trnY and trnfM are present as two identical copies. The G:U and U:N wobbling between the first nucleotide of the anticodon and the third nucleotide of the codon permit the 27 tRNA identified species to translate almost all codons. However, at least two additional tRNA genes, trnl-GAU for AUY codon and trnT-UGU for ACR codon, are required to read all codons used in the liverwort mitochondrial genome. All of the identified tRNA genes are 'native' in liverwort mitochondria, not 'chloroplast-like' tRNAs as are found in the mitochondria of higher plants. This result implies that the tRNA gene transfer from chloroplast to mitochondrial genome in higher plants has occurred after the divergence from bryophytes.     

Two tRNA gene clusters associated with rRNA operons rrnD and rrnE in Bacillus subtilis.

Sequence analysis of cloned rescued DNA fragments from a Bacillus subtilis strain with an inserted recombinant plasmid in ribosomal operon rrnE revealed the presence of two tRNA genes for Met and Asp at the 3' end of the operon. Probing chromosomal DNA from a strain carrying a plasmid inserted in rrnD with a fragment containing the genetically unassigned cluster of 16 tRNA genes revealed that the cluster is located immediately following the rrnD operon. Our findings show that all 10 rrn operons in B. subtilis are associated with tRNA gene clusters.     

tRNA genes and the genetic code

The genetic code describes translational assignments between codons and amino acids. tRNAs and aminoacyl-tRNA synthetases (aaRSs) are those molecules by means of which these assignments are established. Any aaRS recognizes its tRNAs according to some of their nucleotides called identity elements (IEs). Let a 1Mut-similarity Sim (1Mut) be the average similarity between such tRNA genes whose codons differ by one point mutation. We showed that: (1) a global maximum of Sim (1Mut) is reached at the standard genetic code 27 times for 4 sets of IEs of tRNA genes of eukaryotic species, while it is so only 5 times for similarities Sim (C&R) between all tRNA genes whose codons lie in the same column or row of the code. Therefore, point mutations of anticodons were tested by nature to recruit tRNAs from one isoaccepting group to another, (2) because plain similarities Sim (all) between tRNA genes of species within any of the three domains of life are higher than between tRNA genes of species belonging to different domains, tRNA genes retained information about early evolution of cells, (3) we searched the order of tRNAs in which they were most probably assigned to their codons and amino acids. The beginning Ala, (Val), Pro, Ile, Lys, Arg, Trp, Met, Asp, Cys, (Ser) of our resulting chronology lies under a plateau on a graph of Sim (1Mut,IE)(univ.ancestors) plotted over this chronology for a set S(IE) of all IEs of tRNA genes, whose universal ancestors were separately computed for each codon. This plateau has remained preserved along the whole line of evolution of the code and is consistent with observations of Ribas de Pouplana and Schimmel [2001. Aminoacy1-tRNA synthetases: potential markers of genetic code development. Trends Biochem. Sci. 26, 591-598] that specific pairs of aaRSs-one from each of their two classes-can be docked simultaneously onto the acceptor stem of tRNA and hence an interaction existed between their ancestors using a reduced code, (4) sharpness of a local maximum of Sim (1Mut) at the standard code is almost 100% along our chronologies.     

Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster

Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), and tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile)and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region- like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region- like sequences has long persisted in snake mtDNAs, possibly since the original insertion of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T psi C arm of snake mitochondrial tRNAs.      

Disrupted tRNA Gene Diversity and Possible Evolutionary Scenarios

The following unusual tRNAs have recently been discovered in the genomes of Archaea and primitive Eukaryota: multiple-intron-containing tRNAs, which have more than one intron; split tRNAs, which are produced from two pieces of RNA transcribed from separate genes; tri-split tRNAs, which are produced from three separate genes; and permuted tRNA, in which the 5′ and 3′ halves are encoded with permuted orientations within a single gene. All these disrupted tRNA genes can form mature contiguous tRNA, which is aminoacylated after processing by cis or trans splicing. The discovery of such tRNA disruptions has raised the question of when and why these complex tRNA processing pathways emerged during the evolution of life. Many previous reports have noted that tRNA genes contain a single intron in the anticodon loop region, a feature common throughout all three domains of life, suggesting an ancient trait of the last universal common ancestor. In this context, these unique tRNA disruptions recently found only in Archaea and primitive Eukaryota provide new insight into the origin and evolution of tRNA genes, encouraging further research in this field. In this paper, we summarize the phylogeny, structure, and processing machinery of all known types of disrupted tRNAs and discuss possible evolutionary scenarios for these tRNA genes.