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PubMed1997

Ribosomal RNA (rrn) operons in Streptococcus mutans and nucleotide sequence of tRNA(Pro) gene associated with rrnB.

Using a cloned 0.5-kb probe containing an internal fragment of 23S ribosomal RNA from the rrnB operon of Streptococcus mutans, we analyzed various endonuclease digests of the chromosomal DNA isolated from human-derived strains of mutans streptococci. Thus far, the examined S. mutans strains exhibited five ribosomal operons. Here, we describe a ribotyping technique for S. mutans based on restriction and Southern blot analyses with the biotin-labeled homologous probe and chemiluminescence detection. We cloned and sequenced a unique gene encoding tRNA(Pro) downstream from 23S rRNA gene at the 3' end of the operon. Primers designed to the 3' end of the rrnB operon PCR-amplified a 2.3-kb DNA fragment in all tested strains. Restriction fragment length polymorphism analysis of the amplicon revealed a diversity of the single locus among S. mutans isolates, thus establishing a potential use of the technique for the molecular epidemiology of mutans streptococci.

Base Sequence↗

PubMed1993

Deficiency of 1-methylguanosine in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation.

The trmD gene encodes the tRNA(m1G37)methyltransferase, which methylates guanosine (G) to 1-methylguanosine (m1G) at position 37 of tRNAs that read CUN (leucine), CCN (proline), and CGG (arginine) codons. A mutant, trmD3, has previously been isolated, which at high temperature lacks m1G in tRNA, and this deficiency was correlated with a +1 frameshifting activity. In this study, the mechanism of this trmD3-induced frameshift involving mutant tRNA(Pro) and tRNA(Leu) species has been investigated. Potential frameshifting sites for proline tRNAs, CCC-N, were efficiently suppressed in the mutant strain. Hybrid beta-galactosidases encoded by plasmid constructs containing the sites CCC-U and CCC-A were subjected to amino-terminal sequencing. The protein sequences demonstrated that a quadruplet translocation had occurred and that a proline was inserted at these sites, suggesting that a tRNA(Pro) deficient in m1G is the frameshifting agent. Therefore, a mechanism involving a quadruplet codon-anticodon interaction is favoured for trmD3-dependent +1 frameshifting. Of the four potential sites for tRNA(Leu) (CCU-N), two, CCU-U and CCU-C, were significantly suppressed in the trmD3 mutant. Thus, species of tRNA(Leu) may also act as +1 frameshift suppressors. No -1 frameshifting activity was found with the trmD3 mutant.

Amino Acid Sequence↗

PubMed1989

Gene arrangement in sea star mitochondrial DNA demonstrates a major inversion event during echinoderm evolution.

The mitochondrial (mt) DNA from the sea star Pisaster ochraceus has been isolated, restriction-mapped, and cloned into plasmid vectors. Both ribosomal RNA genes, the genes for 12 of the 13 mitochondrial proteins, and 11 of the tRNA genes have been localized by DNA sequence analyses. The sequence arrangement of the genes is markedly different from that seen in sea urchin mitochondrial DNA. A segment of the DNA molecule extending from tRNA(pro), including the tRNA cluster, ND1, ND2, and 16S genes, is inverted in relation to the sea urchin genome. The resulting gene order in the sea star is 12S, 16S, ND2, tRNA cluster, COI. As a result of the inversion, the transcriptional polarity of ND1, ND2, and 16S genes are opposite to that of the 12S and COI genes. The arrangement and transcriptional polarity of the other genes mapped here is the same as seen in urchin.

Animals↗

PubMed1996

A new antisense tRNA construct for the genetic treatment of human immunodeficiency virus type 1 infection.

Different strategies proposed in the literature to attempt gene therapy of AIDS are based mainly on the intracellular production of RNA and protein therapeutics. This report describes the construction and the anti-human immunodeficiency virus type 1 (HIV-1) activity of a new type of antisense tRNA directed against a nucleotide region in the first coding exon of HIV-1 tat (nucleotides 5924 to 5943; Los Alamos data bank) which is conserved among many HIV-1 clones. The anti-tat antisense sequence was inserted into a tRNA(Pro) backbone by replacement of the anticodon loop, without altering the tRNA canonic tetraloop structure. The antisense tRNA was able to interact effectively with its target in vitro. Jurkat cells that constitutively expressed the anti-tat tRNA following retroviral vector transduction exhibited significant resistance to HIV-1 de novo infection. Resistance seemed to correlate with the level of antisense expression. This is the first time that such a tRNA antisense strategy has been shown to be effective as a genetic treatment of HIV-1 infection in tissue culture. The construct design proposed in this report has some intrinsic advantages: the transcript is driven by a polymerase III promoter, the short length of the RNA minimizes effects of intramolecular base pairing that may impair target recognition, and the antisense RNA has the stability and intracellular fate of a native tRNA molecule.

Base Sequence↗

PubMed2005

Association between Archaeal prolyl- and leucyl-tRNA synthetases enhances tRNA(Pro) aminoacylation.

Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA(Pro) charging showed that the catalytic efficiency (k(cat)/K(m)) of ProRS increased 5-fold in the complex with LeuRS compared with the free enzyme, whereas the K(m) for proline was unchanged. No significant changes in the steady-state kinetics of LeuRS aminoacylation were observed upon the addition of ProRS. These findings indicate that ProRS and LeuRS associate in M. thermautotrophicus and suggest that this interaction contributes to translational fidelity by enhancing tRNA aminoacylation by ProRS.

Amino Acyl-tRNA Synthetases↗

PubMed1993

A tRNA gene transcription initiation site is similar to mRNA and rRNA promoters in plant mitochondria.

The gene for tRNA(Phe) is located 292 nucleotides upstream of the tRNA(Pro) gene in the Oenothera mitochondrial genome. Hybridization with in vitro capped primary transcripts indicates a transcription initiation site in the 5' region of the gene for tRNA(Phe). Primer extension experiments show the presence of precursor transcripts covering tRNA(Phe) and adjacent sequences up to a transcription initiation site 181 or 180 nucleotides upstream of the tRNA gene. The genomic sequence at this transcription initiation site contains the consensus motif derived for putative promoters of mitochondrial protein and rRNA coding genes in dicotyledonous plants. This sequence similarity suggests that tRNAs, rRNAs and mRNAs can be transcribed from homologous promoters in plant mitochondria.

Base Sequence↗

PubMed2002

Prevention of mis-aminoacylation of a dual-specificity aminoacyl-tRNA synthetase.

Accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetase is essential for the fidelity of protein synthesis. For Methanococcus jannaschii tRNA(Pro), accuracy is difficult because the cognate prolyl-tRNA synthetase also recognizes and aminoacylates tRNA(Cys) with cysteine. We show here that the unmodified transcript of M. jannaschii tRNA(Pro) is indeed mis-acylated with cysteine. However, the origin of mis-charging is not at the anticodon or acceptor stem, the two hotspots for tRNA(Pro) and tRNA(Cys) identity determinants. Instead, replacement of the D loop in the tRNA core with that of tRNA(Cys) suppresses mis-charging with cysteine without compromising the activity of aminoacylation with proline. The reduced level of cysteine activity of the chimera is not due an editing response of the synthetase and is consistent with a relaxed sensitivity of the tRNA to the analog thiaproline in aminoacylation with cysteine. We suggest that mis-acylation is not due to the presence of cysteine determinants, but to a mis-placed 3' end into the cysteine catalytic site that activates and transfers cysteine to the tRNA. Prevention of mis-placement by alteration of the core structure or by nucleotide modifications in the tRNA illustrates a novel strategy of the dual-specificity synthetase.

Acylation↗

PubMed1994

Clinical and morphologic features of a myopathy associated with a point mutation in the mitochondrial tRNA(Pro) gene.

We studied a 9-year-old girl with progressive weakness of her extremities for two years. Her neurologic evaluation showed weakness of proximal muscles but no ophthalmoparesis. With the exception of elevated serum lactic acid, the general blood screen, EMG, nerve conduction velocity tests, and ECG were normal. Light and electron microscopy of a muscle biopsy showed proliferation of mitochondria containing disorganized cristae. Activities of respiratory chain enzymes containing mitochondrial DNA (mtDNA)-encoded subunits were significantly impaired in muscle homogenates. A G-->A transition at position 15990 previously detected in this patient's muscle was not present in peripheral blood cells of her mother or sister. However, the patient's WBCs appeared to contain a very small percentage of mutant mtDNAs, indicating that the mutation may have originated during early embryogenesis.

Amino Acid Sequence↗

PubMed1994

A new gene encoding tRNA(Pro) (GGG) is present in the chloroplast genome of black pine: a compilation of 32 tRNA genes from black pine chloroplasts.

The chloroplast genome of black pine (Pinus thunbergii), a gymnosperm, contains 32 different tRNA genes, 30 of which correspond to those previously identified in tobacco and rice chloroplast genomes. Two additional genes encode tRNA(Pro) (GGG) and tRNA(Arg) (CCG); the former is newly identified while the latter is present in liverwort, Physcomitrella patens and Angiopteris lygodiifolia, chloroplast genomes. Moreover, a partial copy of the split tRNA(Gly) (UCC) gene and full copies of tRNA(His) (GUG), tRNA(Thr) (GGU) and tRNA(Ser) (GCU) genes are present in the large single-copy region of the genome, suggesting extensive rearrangements of the chloroplast genome during evolution. No tRNA genes whose tRNA products can recognize codons CUU/C (Leu) and GCU/C (Ala) have been found. We propose that the 32 tRNAs are sufficient to read all the 61 sense codons in the black pine system using the "two-out-of-three" and the "U:N wobble" mechanisms.

Base Sequence↗

PubMed2000

Improved crystals of Thermus thermophilus prolyl-tRNA synthetase complexed with cognate tRNA obtained by crystallization from precipitate.

The complex between Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) and its cognate tRNA has been crystallized using two different isoacceptors of tRNA(Pro). Similar bipyramidal crystals of the complexes of ProRSTT with the two different tRNA(Pro) isoacceptors grow within two weeks from 32% saturated ammonium sulfate solution. They belong to space group P4(3)2(1)2, with unit-cell parameters a = 143.1, b = 143.1, c = 228.6 A. The crystals diffract weakly to a maximum resolution of 3.1 A. Superior quality crystals were obtained by growing slowly from precipitate over 5-6 months. These are of the same space group but have slightly altered unit-cell parameters, a = 140.8, b = 140.8, c = 237.0 A. These crystals diffract more strongly to at least 2.8 A resolution and a complete data set to 2.85 A resolution has been collected from a single crystal. Comparison of the packing in the two crystal forms shows that domain flexibility contributes to the presence of different crystal contacts in the two forms.

Amino Acyl-tRNA Synthetases↗

PubMed2002

A cystic fibrosis patient with two novel mutations in mitochondrial DNA: mild disease led to delayed diagnosis of both disorders.

A 21-year-old woman who has been suspected of mitochondrial cytopathy, but negative for common mitochondrial DNA (mtDNA) point mutations and deletions, was screened for unknown mutations in the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE). Her asymptomatic mother's blood DNA was also analyzed and used as a reference. Two tRNA regions showing different TTGE patterns between the proband and her mother were sequenced. Two novel mutations, G15995A in tRNA(pro) and A8326G in tRNA(lys), were revealed. These mutations are present in heteroplasmic states. They both occurred at a nucleotide position that is highly conserved throughout evolution. This patient is also a compound heterozygote for the cystic fibrosis (CF) mutations, DeltaF508 and R347P. The phenotype for R347P has been associated with mild disease. Due to the mild features of the R347P mutation in the CF transmembrane conductance regulator (CFTR) gene and the heterogeneous clinical presentation of the mtDNA disease, the patient was not definitively diagnosed until age 21. This case underscores the importance of a complete mutational analysis of the entire mitochondrial genome when a patient suspected of mitochondrial disorder is negative for common mtDNA mutations.

Adult↗

PubMed2001

Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase.

Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon. The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA(Pro) possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA(Pro) by M. jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem. However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases. Although the human enzyme places a stronger emphasis on G35, the M. jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M. jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like. This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine.

Amino Acid Sequence↗

PubMed1991

Absolute in vivo translation rates of individual codons in Escherichia coli. The two glutamic acid codons GAA and GAG are translated with a threefold difference in rate.

We have determined the absolute translation rates for four individual codons in Escherichia coli. We used our previously described system for direct measurements of in vivo translation rates using small, in-frame inserts in the lacZ gene. The inserts consisted of multiple synthetic 30 base-pair DNA oligomers with high densities of the four individual codons, GAA (Glu), GAG (Glu), CCG (Pro) and CGA (Arg). Our method is independent of expression level, of mRNA half-life and of transcription rate. Codon GAA was found to be translated with a rate of 21.6 codons/second whereas codon GAG was translated 3.4-fold slower (6.4 codons/s). These two codons are read by the same tRNA species. Codon CCG and CGA are both read by abundant tRNA species but nevertheless we found them to be translated slowly with rates of 5.8 and 4.2 codons/second, respectively. The context of these codons were varied, but we found no significant influence of context on their translation rates and we suggest a mechanism for why context may not affect translation rates. One insert with a low translation rate gave results that most readily can be explained by assuming queue formation of ribosomes on the insert. Such a queue was found to reduce the expression level by approximately 35%. Our experiments allowed us to estimate the average distance between ribosomes and thereby the translation initiation frequency on the wild-type lacZ mRNA. This was found to be one per three seconds.

Base Sequence↗

PubMed2002

Imbalance of tRNA(Pro) isoacceptors induces +1 frameshifting at near-cognate codons.

Increased expression of the CCU/CCA/CCG-decoding tRNA(Pr)(o)3 on a multicopy plasmid leads to suppression of several +1 frameshift mutations in Salmonella enterica serovar Typhimurium. Systematic analysis of the site of frameshifting indicates that excess tRNA(Pr)(o)3 promotes near-cognate decoding at CCC codons. Re-phasing of the reading frame can be achieved by a subsequent slippage of the tRNA onto a cognate codon in the +1 reading frame. Frameshifting appears to be due to an imbalance of CCC-cognate and near-cognate tRNAs, as the effect of excess tRNA(Pr)(o)3 on reading frame maintenance can be reversed by increasing simultaneously the concentration of the cognate tRNA(Pr)(o)2. Finally, the cmo5U modification present at position 34 of tRNA(Pr)(o)3, which allows this tRNA to decode CCU in addition to CCG and CCA, also affects frameshifting, indicating that the ability of the near-cognate tRNA to decode a cognate codon efficiently in the alternative reading frame is important for re-phasing of the reading frame.

Alcohol Oxidoreductases↗

PubMed1987

Isolation of three kinds of human endogenous retrovirus-like sequences using tRNA(Pro) as a probe.

Three kinds of human endogenous retrovirus-like sequences (HuERS-P1, 2 and 3) were isolated from a HeLa cell genomic library using the 3'-half fragment of proline tRNA as a hybridization probe. These elements contained putative primer binding sites complementary to the 3'-terminus of proline tRNA and long terminal repeats (LTRs) characteristic of retrovirus provirus. The LTR sequence of HuERS-P1 consisted of about 690 nucleotides and contained a CAT box, a TATA box and a polyadenylation signal. A complete unit of an Alu family sequence was inserted into the 5'-LTR of one of the clones. HuERS-P2 also contained a TATA box and a polyadenylation signal in its LTR (about 840 nucleotides long), but the LTR sequence of this element was quite different from that of HuERS-P1. Although clone HuERS-P3 contained only the 5'-LTR region, this LTR sequence contained a CAT box, a TATA box and a poly-adenylation signal and was quite similar to the LTR sequence of the recently isolated human retrovirus-related sequence HuRRS-P (Kröger, B. and Horak, I. (1987) J. Virol., 61, 2071-2075). Human and simian DNAs contain 10 to 40 copies of these elements, but mouse DNA does not contain these elements.

Base Sequence↗

PubMed1999

Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity.

In Salmonella typhimurium, the tRNA(m1G37)methyltransferase (the product of the trmD gene) catalyzes the formation of m1G37, which is present adjacent and 3' of the anticodon (position 37) in seven tRNA species, two of which are tRNA(Pro)CGG and tRN(Pro)GGG. These two tRNA species also exist as +1 frameshift suppressor sufA6 and sufB2, respectively, both having an extra G in the anticodon loop next to and 3' of m1G37. The wild-type form of the tRNA(m1G37)methyltransferase efficiently methylates these mutant tRNAs. We have characterized one class of mutant forms of the tRNA(m1G37)methyltransferase that does not methylate the sufA6 tRNA and thereby induce extensive frameshifting resulting in a nonviable cell. Accordingly, pseudorevertants of strains containing such a mutated trmD allele in conjunction with the sufA6 allele had reduced frameshifting activity caused by either a 9-nt duplication in the sufA6tRNA or a deletion of its structural gene, or by an increased level of m1G37 in the sufA6tRNA. However, the sufB2 tRNA as well as the wild-type counterparts of these two tRNAs are efficiently methylated by this class of structural altered tRNA(m1G37)methyltransferase. Two other mutations (trmD3, trmD10) were found to reduce the methylation of all potential tRNA substrates and therefore primarily affect the catalytic activity of the enzyme. We conclude that all mutations except two (trmD3 and trmD10) do not primarily affect the catalytic activity, but rather the substrate specificity of the tRNA, because, unlike the wild-type form of the enzyme, they recognize and methylate the wild-type but not an altered form of a tRNA. Moreover, we show that the TrmD peptide is present in catalytic excess in the cell.

Amino Acid Sequence↗

PubMed1994

Patterns of sequence variation in the mitochondrial D-loop region of shrews.

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D-loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.

Animals↗

PubMed1994

Sequence evolution and phylogenetic signal in control-region and cytochrome b sequences of rainbow fishes (Melanotaeniidae).

The nucleotide sequences of segments of the cytochrome b gene (351 bp), the tRNA(Pro) gene (49 bp), and the control region (approximately 313 bp) of mitochondrial DNA were obtained from 26 fish representing different populations and species of Melanotaenia and one species of Glossolepis, freshwater rainbow fishes confined to Australia and New Guinea. The purpose was to investigate relative rates and patterns of sequence evolution. Overall levels of divergence were similar for the cytochrome b and tRNA control-region sequences, both ranging from < 1% within subspecies to 15%-19% between genera. However, the patterns of sequence evolution differed. For the cytochrome b gene, transitions consistently exceeded transversions, the bias ranging from 4.2:1 to 2:1, depending on the level of sequence divergence. However, in the control-region sequence, a bias toward transitions (2:1) was observed only in comparisons between very similar sequences, and transversions outnumbered transitions in comparisons of divergent sequences. Graphic comparisons suggested that the control region was saturated for transitions at relatively low levels of sequence divergence but accumulated transversions at a greater rate than did the cytochrome b sequence. These distinct patterns of base substitution are associated with differences in A+T content, which is 70% for the tRNA control-region segment versus 50% for cytochrome b. A test for skewness in the distribution of lengths of random trees indicated that both segments contained phylogenetic signal. Parsimony analyses of the data from the two regions, with or without weighting schemes appropriate to the respective patterns of sequence evolution, identified the same five groupings of sequences, but the relationships among the groups differed. However, in most cases the branches uniting different combinations of groups were poorly supported, and the differences among topologies were insignificant. Considering the observed patterns of base substitution and the results of the phylogenetic analyses, we deduce that both the control region and cytochrome b are appropriate for population genetic studies but that the control region is less effective than cytochrome b for resolving relationships among divergent lineages of rainbow fishes.

Animals↗