Sequence analysis of Saccharomyces exiguus mitochondrial DNA reveals an RNase P RNA gene flanked by two tRNA genes.
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Base Composition↗
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Effect of a mutation in the anticodon of human mitochondrial tRNAPro on its post-transcriptional modification pattern.
Although the gene sequences of all 22 tRNAs encoded in the human mitochondrial genome are known, little information exists about their sequences at the RNA level. This becomes a crucial limitation when searching for a molecular understanding of the growing number of maternally inherited human diseases correlated with point mutations in tRNA genes. Here we describe the sequence of human mt-tRNAPropurified from placenta. It shows absence of editing events in this tRNA and highlights the presence of eight post-transcriptional modifications. These include T54, never found so far in an animal mt-tRNA, and m1G37, a modification known to have fundamental functional properties in a number of canonical tRNAs. Occurrence of m1G37 was further investigated in an analysis of the substrate properties of in vitro transcripts of human mt-tRNAProtowards pure Escherichia coli methylguanosine transferase. This enzyme properly methylates G37 in mt-tRNA and is sensitive to the presence of a second G at position 36, neighboring the target nucleotide for methylation. Since mutation of nt 36 was shown to be correlated with myopathy, the potential consequences of non-modification or under-modification of mt-tRNA nucleotides in expression of the particular myopathy and of mitochondrial diseases in general are discussed.
Anticodon↗
The use of sequence comparison to detect 'identities' in tRNA genes.
We have developed a computational method that detects 'identities' in tRNA genes by using principal component analysis to classify the sequences of bases in tRNA genes into groups of similar sequences and then comparing the distribution of sequences of bases, in order to extract characteristic bases that are conserved within a group but differ between groups. These classification and comparison procedures are applied recursively to classify the sequences into hierarchical groups, so that multiple levels of characteristic sites can be detected. By using this computational method, we were able to detect many characteristic sites in the T and D domains of tRNAs, as well as the characteristic sites that had already been detected experimentally. This suggests that bases not only in the contact regions but also in the elbow regions, which determine the structure and dynamics of the whole tRNA molecule, are important to the tRNA-aminoacyl tRNA synthetase recognition.
Amino Acyl-tRNA Synthetases↗
Screening by tRNA primer extension analysis of porcine kidney mRNA libraries defines a novel endogenous porcine retroviral long terminal repeat.
BACKGROUND: We have taken advantage of the common requirement of all eukaryotic retroelements for a specific tRNA primer to initiate DNA synthesis and applied a previously described in vitro screening methodology to the analysis of in vivo porcine tissues for transcriptionally active retroviral sequences. METHODS: A series of 18-base pair (bp) 3' tRNA oligomers complementary to established primer binding sites for a variety of vertebrate retroviruses, retrotransposons, and retroposons were applied to primer extension analysis of kidney poly(A) mRNA. Primer extension products are predicted to represent "strong stop" signals characteristic of the initial stages of retroviral transcription. RESULTS: Several extension products were cloned, sequenced, and analyzed as probes for screening the porcine genome for potentially active retroviral sequences. We used this strategy to identify and clone a 655-bp 5' long terminal repeat of a porcine retrovirus with significant homology to the simian sarcoma virus. This transcriptionally active virus has an 82-bp U5 region, a conserved AATAAA polyadenylation sequence, a 39-bp repeat reminiscent of other retroviral enhancers, and a unique glycine primer binding site. CONCLUSION: Our results suggest that tRNA primer cloning can effectively identify novel retroviral elements.
Animals↗
Nucleotide sequence of a Lophopyrum elongatum mitochondrial proline tRNA gene.
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DNA, Mitochondrial↗
Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine.
The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.
Anticodon↗
Editing of transfer RNAs in Acanthamoeba castellanii mitochondria.
With the discovery of RNA editing, a process whereby the primary sequence of RNA is altered after transcription, traditional concepts of genetic information transfer had to be revised. The known RNA editing systems act mainly on messenger RNAs, introducing sequence changes that alter their coding properties. An editing system that acts on transfer RNAs is described here. In the mitochondria of Acanthamoeba castellanii, an amoeboid protozoan, certain transfer RNAs differ in sequence from the genes that encode them. The changes consist of single-nucleotide conversions (U to A, U to G, and A to G) that appear to arise posttranscriptionally, are localized in the acceptor stem, and have the effect of correcting mismatched base pairs. Editing thus restores the base pairing expected of a normal transfer RNA in this region.
Acanthamoeba↗
Expression of a yeast intron-containing tRNA in the archaeon Haloferax volcanii.
Expression of the yeast tRNAPro(UGG) gene in Haloferax volcanii resulted in the production of a single stable transcript that had not undergone intron processing or processing of 5' and 3' flanking sequences. Mutation of the exon-intron boundary region of this RNA to produce a precursor RNA with the preferred halobacterial consensus exon-intron boundary structure did not restore intron processing. Processing of 5' and 3' flanking sequences was restored when the acceptor stem U6-U67 pair was changed to A6-U67. The significance of these results in defining the recognition requirements of tRNA maturation enzymes in the halophilic domain Archaea is discussed.
Base Sequence↗
ihfA gene of the bacterium Myxococcus xanthus and its role in activation of carotenoid genes by blue light.
Myxococcus xanthus responds to blue light by producing carotenoids. Several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes. We had already reported the isolation of a carotenoid-less, Tn5-induced strain (MR508), whose mutant site was unlinked to the indicated regulatory genes. Here, we show that OmegaMR508::Tn5 affects all known light-inducible promoters in different ways. It blocks the activation of two of them by light but makes the activity of a third one light independent. The OmegaMR508 locus has been cloned and sequenced. The mutation had occurred at the promoter of a gene we propose is the M. xanthus ortholog of ihfA. This encodes the alpha subunit of the histone-like integration host factor protein. An in-frame deletion within ihfA causes the same effects as the OmegaMR508::Tn5 insertion. Like other IhfA proteins, the deduced amino acid sequence of M. xanthus IhfA shows much similarity to HU, another histone-like protein. Sequence comparison data, however, and the finding that the M. xanthus gene is preceded by gene pheT, as happens in other gram-negative bacteria, strongly argue for the proposed orthology relationship. The M. xanthus ihfA gene shows some unusual features, both from structural and physiological points of view. In particular, the protein is predicted to have a unique, long acidic extension at the carboxyl terminus, and it appears to be necessary for normal cell growth and even vital for a certain wild-type strain of M. xanthus.
Amino Acid Sequence↗
Site-specific integrative elements of rhizobiophage 16-3 can integrate into proline tRNA (CGG) genes in different bacterial genera.
The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R. meliloti 1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously. The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA.
Bacteriophages↗
Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei.
Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed phiE125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of phiE125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with phiE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-host-range plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to phiE125. The 53,373-bp phiE125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene. While the overall genetic organization of the phiE125 genome was similar to lambda-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The phiE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that phiE125 is a new member of the lambda supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei.
Burkholderia↗
Sequence requirements for removal of tRNA by an isolated human immunodeficiency virus type 1 RNase H domain.
Retroviral reverse transcriptase-associated RNase H enzymes are responsible for degradation of viral RNA, including removal of the tRNA primer after plus-strand strong-stop synthesis and cleavage of the polypurine tract primer. These activities are required for the complex viral replication and result in generation of the long terminal repeats. The human immunodeficiency virus type 1 (HIV-1) RNase H domain has been expressed independently of the polymerase domain and possesses Mn2+-dependent activity with a hexahistidine tag. The isolated domain maintains the ability to specifically remove a tRNA primer mimic. In this study, the substrate determinants for recognition of the cognate tRNA3Lys are defined. Model substrates were constructed which mimic the RNA-DNA hybrid obtained from plus-strand strong-stop synthesis. Deletion substrates containing only 12, 9, or 6 positions of the tRNA primer were capable of being cleaved by the isolated RNase H domain. Mismatch and bromodeoxyuridine mutagenesis analysis indicated that positions 2, 3, 4, and 6, when mutated, affected the specificity of RNase H activity. Substitution substrates indicated that positions 4 and 6 within the RNA primer were important for recognition and cleavage by the HIV-1 isolated RNase H domain. Moloney murine leukemia virus-HIV-1 hybrid substrates were constructed which demonstrated that changes to HIV-1 sequences at positions 4 and 6 were sufficient but not optimal for regaining cleavage by the isolated HIV-1 RNase H domain. Optimal site-specific cleavage between the terminal ribonucleotide A and ribonucleotide C requires additional sequences beyond the first six positions but less than nine.
Binding Sites↗
Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.
To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGG from Saccharomyces cerevisiae was altered by site-directed mutagenesis of the suf8 gene. Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39). The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction. The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G.U interactions. However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision. We also determined the extent to which the suf8 alleles produce functional products in vivo. Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation. The formation of a functional product was assayed by the ability of each allele to suppress the +1 frameshift mutation his4-713 through four-base codon reading, as shown previously for the SUF8-1 suppressor allele. We found that alleles containing any standard base pair or G.U pair at position 31/39 in the anticodon stem failed to suppress his4-713. We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon. However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression. Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppression alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro. These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affects tRNA splicing.
Anticodon↗
In vivo collagen turnover during development of thyroxine-induced left ventricular hypertrophy.
Cardiac fibroblasts synthesize large amounts of procollagens, yet only a small fraction of mature collagens accumulate in the extracellular matrix. To determine the roles of intracellular degradation of newly synthesized procollagens and extracellular degradation of mature collagens during normal growth and during thyroxine-induced left ventricular hypertrophy, in vivo left ventricular procollagen synthetic rates were assessed in control rats and rats treated with L-thyroxine for 1, 2, 4, and 8 wk (1 mg.kg-1.day-1). A modification of the flooding infusion method was developed using measurements of cardiac prolyl-tRNA, and tissue-free and protein-bound hydroxyproline specific radioactivities 60 min after intravenous administration of a massive dose of [3H]proline. Degradative rates of newly synthesized procollagens and mature collagens were then derived as the difference between rates of procollagen synthesis and collagen accumulation. Left ventricular procollagen synthetic rates were markedly increased after 1 wk of hormone administration (256 +/- 16 and 166 +/- 13 micrograms/day per left ventricle for thyroxine-treated and control animals, respectively; P less than 0.01). An even greater increase in procollagen synthetic rates was observed after 8 wk (438 +/- 46 and 202 +/- 18 micrograms/day for thyroxine-treated and control animals, respectively; P less than 0.01). Despite increased procollagen synthesis, disproportionate accumulation of fibrillar collagens (assessed as the relative concentration of protein-bound hydroxyproline in left ventricular tissue) did not occur. Derived left ventricular degradative rates for newly synthesized procollagens as well as for mature collagens were increased in thyroxine-treated animals. Increased procollagen synthesis, enhanced flux of newly synthesized procollagens through intracellular degradative pathways, and extensive extracellular matrix remodeling without disproportionate collagen accumulation are characteristics of this form of "physiological" left ventricular hypertrophy.
Animals↗
Regional localization of loci on chromosome 14 using somatic cell hybrids.
We have used a panel of human x rodent somatic cell hybrids containing translocations involving chromosome 14 to regionally localize 17 genes and 5 random segments previously mapped to chromosome 14. Each hybrid cell line contains a specific fragment of chromosome 14, with breakpoints at 14q11.2, 14q21, 14q22, 14q24.3 or in two different regions of 14q32.1. The enzyme deficient in glycogen storage disease type VI, liver glycogen phosphorylase (PYGL), has been localized by in situ hybridization to 14q21-->q22, near the q21-->q22 band interface. Four additional genes, chromogranin A (CHGA), myosin (MYH7), tRNA proline 2 (TRP2) and c-FOS (FOS) and four random segments, D14S26, D14S12, D14S14 and D14S13 have been more precisely localized. This study also defines a hybrid cell panel with seven mapping intervals, that will be useful for further physical mapping of new loci.
Animals↗
The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo.
In Salmonella enterica serovar Typhimurium five of the eight family codon boxes are decoded by a tRNA having the modified nucleoside uridine-5-oxyacetic acid (cmo5U) as a wobble nucleoside present in position 34 of the tRNA. In the proline family codon box, one (tRNAProcmo5UGG) of the three tRNAs that reads the four proline codons has cmo5U34. According to theoretical predictions and several results obtained in vitro, cmo5U34 should base pair with A, G, and U in the third position of the codon but not with C. To analyze the function of cmo5U34 in tRNAProcmo5UGG in vivo, we first identified two genes (cmoA and cmoB) involved in the synthesis of cmo5U34. The null mutation cmoB2 results in tRNA having 5-hydroxyuridine (ho5U34) instead of cmo5U34, whereas the null mutation cmoA1 results in the accumulation of 5-methoxyuridine (mo5U34) and ho5U34 in tRNA. The results suggest that the synthesis of cmo5U34 occurs as follows: U34 -->(?) ho5U -->(CmoB) mo5U -->(CmoA?) cmo5U. We introduced the cmoA1 or the cmoB2 null mutations into a strain that only had tRNAProcmo5UGG and thus lacked the other two proline-specific tRNAs normally present in the cell. From analysis of growth rates of various strains and of the frequency of +1 frameshifting at a CCC-U site we conclude: (1) unexpectedly, tRNAProcmo5UGG is able to read all four proline codons; (2) the presence of ho5U34 instead of cmo5U34 in this tRNA reduces the efficiency with which it reads all four codons; and (3) the fully modified nucleoside is especially important for reading proline codons ending with U or C.
Amino Acid Sequence↗
Stimulation of collagen synthesis in fibroblast cultures by a triterpene extracted from Centella asiatica.
The drug "Titrated Extract from Centella asiatica" (TECA), used for its stimulating properties on the healing of wounds, is a mixture of 3 terpenes extracted from a tropical plant: asiatic acid (30%, w/w), madecassic acid (30%, w/w) and asiaticoside (40%, w/w). The effects of TECA and its individual components were checked on human foreskin fibroblast monolayer cultures. TECA increased the collagen synthesis in a dose-dependent fashion whereas a simultaneous decrease in the specific activity of neosynthesized collagen was observed. Asiatic acid was found to be the only component responsible for collagen synthesis stimulation. TECA and all three terpenes increased the intracellular free proline pool. This effect was independent of the stimulation of collagen synthesis.
Cell Count↗
Prolyl-tRNA(Pro) in the A-site of SecM-arrested ribosomes inhibits the recruitment of transfer-messenger RNA.
Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3'-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to tRNA(3Gly) in the peptidyl-tRNA binding site, and prolyl-tRNA(2Pro) is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of prolyl-tRNA(2Pro) on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of tRNA(2Pro) readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses.
Binding Sites↗