Sequence of tRNAAsn gene of Saccharomyces cerevisiae.
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Base Sequence↗
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Results for “RNA, Transfer, Asn”
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Expression of small RNAs by Bacillus sp. strain PS3 and B. subtilis cells during sporulation.
A small RNA sequence identified in an rRNA-tRNA cluster from the thermophilic Bacillus sp. strain PS3 was examined. An oligonucleotide probe specific for the RNA bound to multiple restriction fragments in Bacillus sp. strain PS3 DNA, thus several copies of this sequence occur in its genome. Similar findings were observed using DNA from B. subtilis, B. stearothermophilus, Escherichia coli, Staphylococcus aureus, Haemophilus influenzae and Thermus thermophilus. This sequence apparently is widespread in the eubacteria. Northern analysis of RNA from sporulating Bacillus sp. strain PS3 and B. subtilis cells revealed RNA species homologous to the probe in both bacteria. Expression of the small RNA in B. subtilis depended on sigma H.
Bacillus↗
A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes.
We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5Asn genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.
Animals↗
RSEARCH: finding homologs of single structured RNA sequences.
BACKGROUND: For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature. No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure. RESULTS: We have developed a program, RSEARCH, that takes a single RNA sequence with its secondary structure and utilizes a local alignment algorithm to search a database for homologous RNAs. For this purpose, we have developed a series of base pair and single nucleotide substitution matrices for RNA sequences called RIBOSUM matrices. RSEARCH reports the statistical confidence for each hit as well as the structural alignment of the hit. We show several examples in which RSEARCH outperforms the primary sequence search programs BLAST and SSEARCH. The primary drawback of the program is that it is slow. The C code for RSEARCH is freely available from our lab's website. CONCLUSION: RSEARCH outperforms primary sequence programs in finding homologs of structured RNA sequences.
Animals↗
Genomic organization of the human asparagine transfer RNA genes: localization to the U1 RNA gene and class I pseudogene repeat units.
Previously isolated human DNA clones containing asparagine transfer RNA (tRNAAsn) genes have been used to determine the genomic organization of this multigene family in man. One clone also contained a gene for U1 RNA, and so the organization of the two multigene families could be directly compared. The majority, and perhaps all, of the human tRNAAsn genes map to the same chromosome bands as do the U1 RNA true genes and class I pseudogenes located on the short and long arms, respectively, of chromosome 1. These two gene clusters were independently isolated using a somatic-cell hybrid minipanel, and use of repeat-unit DNA polymorphisms showed that one tRNA gene clone maps to the short-arm gene cluster and the other to the long-arm gene cluster. Electron microscopy of heteroduplexes between these two clones showed duplex formation along the proposed region of overlap between them, indicating that the short- and long-arm gene clusters are structurally related. I suggest that the split into two distinct loci was facilitated by a pericentric chromosome inversion. This would have had the effect of positioning the genes currently on the long arm adjacent to the centromeric heterochromatin, perhaps resulting in a "position effect" on transcription of these genes. Restriction fragments of different sizes were found that were common to a majority of repeat units, depending on the restriction enzyme being used. Pulsed-field electrophoresis revealed that fragments of molecular weight of 180 kb were common to each unit (or multiples of units). These fragments also contained U1 RNA gene sequences. I therefore propose that these two gene families are closely linked on repeat units (or multiples of units) of 180 kb in size, which are probably organized in tandem arrays.
Chromosome Inversion↗
Evolution of the WANCY region in amniote mitochondrial DNA.
In most vertebrate mitochondrial genomes, the site for initiation of light-strand replication, OL, is found within a cluster of five transfer RNA (tRNA) genes (tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), and tRNA(Tyr)). This region and part of the adjacent cytochrome c oxydase subunit I (COI) gene were sequenced for two crocodilian, two turtle, and one snake species and for Sphenodon punctatus; part of the adjacent nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene was also sequenced for the crocodilian and turtle species. All had the typical vertebrate gene order. The turtles and the snake have a lengthy noncoding sequence between the tRNA(Asn) and tRNA(Cys) genes that we assumed to be homologous to the mammalian OL. The crocodilians and Sphenodon lack such a sequence, a condition they share with birds. Most proposed phylogenies for the amniotes require that OL at this position was lost at least twice during their diversification or was evolved independently more than once. Within the five tRNA genes, frequencies of substitutions are much higher in loops than in stems. Many loops vary dramatically in size among the species; in the most extreme case, the D-arm of the Sphenodon tRNA(Cys) is a "D-arm replacement" loop of seven nucleotides. Frequency of transitions in stems is relatively uniform across tRNAs, but frequency of transversions varies greatly. Mismatches in stems are infrequent, and their relative frequency in a specific tRNA is unrelated to the frequency of substitution in the corresponding gene. Several features of mammalian mitochondrial tRNAs are conserved in WANCY tRNAs throughout amniotes. The inferred initiation codon for COI is GTG in crocodilians, turtles, and the snake, a condition they share with fishes, certain amphibians, and birds. TTG appears to be the initiation codon for COI in Sphenodon; if correct, this would be a novel initiation codon for vertebrate mitochondrial DNA. Phylogenetic analyses of the inferred amino acid sequences of ND2 and COI support the sister-group relationship of birds and crocodilians and suggest that mammals are an early derived lineage within the amniotes.
Amino Acid Sequence↗
Hypomodification of transfer RNA in cancer with respect to queuosine.
Queuosine is a highly modified nucleoside analogue of guanosine. It is present only in the first position of anticodon loop of specific tRNA i.e., tRNA(his), tRNA(asp), tRNA(asn) and tRNA(tyr) and post transcriptionally modified with base-for-base exchange of guanine to queuine. The transfer RNA modifying enzyme transfer RNA guanine transglycosylase (TGTase) catalyzes the modification of tRNAs. Transfer RNA is completely modified with respect to queuosine in mature tissue, however modification is often incomplete in mitotically active cells. Hypomodification of transfer RNA is correlated with cell proliferation and malignancy. In the present study queuosine modification of transfer RNA and TGTase activity is compared in normal, Dalton's lymphoma ascites transplanted (DLAT) cancerous and queuine treated DLAT cancerous mouse liver. Transfer RNA of cancerous mouse is hypomodified in terms of queuosine modification. TGTase activity of cancerous mouse is found to decrease to less then half of enzyme activity of normal mouse; suggesting that the enzyme may be responsible for transfer RNA hypomodification. Exogenous treatment of queuine during development of cancer improves the queuosine modification of transfer RNA. The activators NaPP and ATP enhance TGTase activity of normal and DLAT cancerous mouse, where as 7mG inhibits the TGTase activity.
Animals↗
Horizontal gene transfer of "prototype" Nramp in bacteria.
Eukaryotic Nramp genes encode divalent metal ion permeases important for nutrition and resistance to microbial infection. Bacterial homologs encode proton-dependent transporters of manganese (MntH), and other divalent metal ions. Bacterial MntH were classified in three homology groups (A, B, C) and MntH C further subdivided in Calpha, Cbeta, Cgamma. The proteins from C. tepidum (MntH B) and E. faecalis (MntH Cbeta1, 2), divergent in sequence and hydropathy profile, conferred increased metal sensitivity when expressed in E. coli, suggesting conservation of divalent metal transport function in MntH B and C. Several genomic evidence suggest horizontal gene transfer (HGT) of mntH C genes: (i) The enterobacteria Wigglesworthia mntH Cbeta gene is linked to an Asn t-RNA, and its sequence most conserved with Gram positive bacteria homologs; (ii) all the Cbeta genes identified in oral streptococcaceae are associated with different potentially mobile DNA elements; (iii) Lactococcus lactis and Burkholderia mallei genomes contain an mntH gene prematurely terminated and a novel full-length mntH C gene; (iv) remarkable sequence relatedness between the unicellular alga C. reinhardtii "prototype" Nramp and some MntH Calpha (e.g., Nostoc spp., Listeria spp.) suggests HGT between Eukarya and Bacteria. Other "prototype" Nramp genes (intronless, encoding proteins strongly conserved with MntH A and B proteins) identified in invertebrates represent a possible source for transfer of Nramp genes toward opportunistic bacteria. This study demonstrates complex evolution of MntH in Bacteria. It is proposed that "prototype" Nramp are ancestors of bacterial MntH C proteins, which could facilitate bacterial infection.
Bacteria↗
Mitochondrial transfer RNA genes from fungi (Aspergillus nidulans) and plants (Lupinus luteus) are transcribed in Xenopus laevis oocyte nuclei.
Three plasmids containing Aspergillus nidulans mitochondrial transfer RNA genes were microinjected into the nucleus of Xenopus laevis oocytes. Plasmids that contained a single tRNACys gene or two tRNA genes (Arg and Asn) yielded tRNA-sized transcripts. Plasmids containing cloned Lupinus mitochondrial tRNA genes were also transcribed and processed in X. laevis nuclei.
Animals↗
Region of a conserved sequence motif in a class II tRNA synthetase needed for transfer of an activated amino acid to an RNA substrate.
The class II Escherichia coli alanine tRNA synthetase aminoacylates RNA miniduplexes, which reconstruct the acceptor end of alanine tRNA with the critical G3:U70 base pair. A benzophenone photoaffinity label attached adjacent to G3:U70 in a miniduplex substrate was previously cross-linked to a long enzyme peptide that begins at Gly161 between the class-defining motifs 2 and 3 [Musier-Forsyth, K., & Schimmel, P. (1994) Biochemistry 33, 773-779]. To identify side chains in this peptide that potentially contribute hydrogen bonding or catalytic determinants for the RNA-dependent step of the aminoacylation reaction, peptide functional side chains that are conserved among sequenced alanine enzymes (Asp, Asn, Arg, Glu, Gln, and Tyr) were individually replaced. Of the 21 mutant proteins so generated, one was identified that was not viable even though it accumulated in vivo. This Asp235-->Ala mutant enzyme is defective in the rate of transfer of the activated amino acid to the 3'-end of the RNA substrate. The conserved Asp235 is at the beginning of motif 3. By comparison with the crystal structure of the related class II yeast aspartate tRNA synthetase complexed with tRNA(Asp) (Cavarelli et al., 1993), we suggest that D235 is not in direct contact with acceptor helix base pairs such as G3:U70. Instead, we propose that D235 contributes to transfer-step interactions at the 3'-end of alanine tRNA. Because D235 in alanine tRNA synthetase is at the beginning of one of the conserved motifs that define class II tRNA synthetases, this region of the structure may in general be important for the transfer step.
Alanine-tRNA Ligase↗
Analysis of transfer RNA during the early embryogenesis of the freshwater teleost, Heteropneustes fossilis.
Total RNA as well as transfer RNA were quantified from mature ova apart from four different embryonic stages namely mid-cleavage, early gastrula, mid-gastrula and organogenesis of the freshwater teleost Heteropneustes fossilis. Total RNA as well as transfer RNA quantity follow a similar variation pattern, being maximum during mid-gastrulation. When analysed by total amino acid acceptance capacity, transfer RNA shows its maximum activity during mid-gastrulation. This coincides with the higher ratio of tRNA to total RNA at this stage. The relative aminoacylation capacity for Ser, Gly, Asn and Thr are found to be higher (9-34%) compared to that for other amino acids. Total tRNA, resolved into three peaks upon HPLC fractionation, shows a high cumulative peak area during mid-gastrulation and organogenesis. These results indicate a switch over of maternal to embryonic translation machinery during gastrulation.
Animals↗
Domain-specific recruitment of amide amino acids for protein synthesis.
The formation of aminoacyl-transfer RNA is a crucial step in ensuring the accuracy of protein synthesis. Despite the central importance of this process in all living organisms, it remains unknown how archaea and some bacteria synthesize Asn-tRNA and Gln-tRNA. These amide aminoacyl-tRNAs can be formed by the direct acylation of tRNA, catalysed by asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, respectively. A separate, indirect pathway involves the formation of mis-acylated Asp-tRNA(Asn) or Glu-tRNA(Gln), and the subsequent amidation of these amino acids while they are bound to tRNA, which is catalysed by amidotransferases. Here we show that all archaea possess an archaea-specific heterodimeric amidotransferase (encoded by gatD and gatE) for Gln-tRNA formation. However, Asn-tRNA synthesis in archaea is divergent: some archaea use asparaginyl-tRNA synthetase, whereas others use a heterotrimeric amidotransferase (encoded by the gatA, gatB and gatC genes). Because bacteria primarily use transamidation, and the eukaryal cytoplasm uses glutaminyl-tRNA synthetase, it appears that the three domains use different mechanisms for Gln-tRNA synthesis; as such, this is the only known step in protein synthesis where all three domains have diverged. Closer inspection of the two amidotransferases reveals that each of them recruited a metabolic enzyme to aid its function; this provides direct evidence for a relationship between amino-acid metabolism and protein biosynthesis.
Amides↗
The 23S-5S spacer of two rRNA loci of Streptococcus salivarius subsp thermophilus includes a promoter.
The nucleotide sequence of the 3' part of a ribosomal and transfer RNA locus from Streptococcus salivarius subsp thermophilus NST1403 was determined. The sequenced DNA fragment includes the 3' end of a 23S rRNA gene, a 5S rRNA gene, a tRNA(asn) gene and a potential transcriptional terminator. The tRNA gene does not encode for the CCA 3'terminus of mature tRNA. We compared this sequence to a promoter-carrying DNA fragment sequence (P20) of Streptococcus salivarius subsp thermophilus A054 [1]. We found that the P20 sequence included the 3' end of a 23S rRNA gene, a 5S rRNA gene and the 5' part of a tRNA(val) gene. The two 23S-5S spacer sequences are identical and contain a promoter and a potential 23S rRNA processing site. Therefore, 5S rRNA and tRNA genes could be transcribed from a promoter located within the 23S-5S spacer of at least two of the six rRNA loci.
Base Sequence↗
The Yersinia high-pathogenicity island (HPI): evolutionary and functional aspects.
The high-pathogenicity island (HPI) is a genomic island essential for the mouse-virulence phenotype in Yersinia and indispensable for pathogenicity of Yersinia and certain pathotypes of Escherichia coli. In contrast to most genomic islands, the HPI is a functional island widely disseminated among members of the family of Enterobacteriaceae. The HPI-encoded phage P4-like integrase together with excisionase and recombination sites make up the genetic mobility module of the island, while the siderophore yersiniabactin biosynthesis and uptake system comprises its functional part with respect to fitness and pathogenicity. The HPI-integrase promotes integration of the island into attB sites represented by three to four asn tDNAs in Yersinia pestis and E. coli. An additional enzyme, excisionase, is essential for efficient excision of the HPI from the initial site of integration. Furthermore a unique type of HPI has been characterized in the E. coli strain ECOR31 carrying a functional conjugative mating pair formation (Mpf) and a DNA-processing system, both of which are characteristic of integrative and conjugative elements (ICE). A model of conjugative transfer for the dissemination of HPIs is proposed in which the excised HPI is mobilized to a new recipient either trapped by a transmissive asn tDNA-carrying plasmid or autonomously as an ICE named ICEEcl.
Animals↗
Identification of the minor guanylated tRNA of rabbit reticulocytes.
Two of the tRNA's found in rabbit reticulocytes are substrates for a post-transcriptional modification leading to the incorporation of guanine into the polynucleotide chain. The major guanylated tRNA was previously identified as tRNA (His). In the present report we show that the minor guanylated tRNA is tRNA (Asn), and that just as in the case of tRNA (His), the guanine is located in an internal position. There are only two tRNA (Asn) in reticulocytes. We further show that one of these, the one that is not labeled with guanine, contains the hypermodified base known as Q. tRNA (Asn) does not contain Q.
Animals↗
Structure and organization of a cluster of sic tRNA genes in the space between tandem ribosomal RNA gene sets in Bacillus subtilis.
Hybridization of Southern blots of EcoRI digests of total Bacillus subtilis DNA with ribosomal RNA and transfer RNA probes provides evidence of highly clustered tRNA and rRNA genes, with several tRNA clusters being located in spaces which span between tandem ribosomal RNA gene sets. Clones containing these tRNA clusters were isolated from a Charon 4A library. One of them, denoted trrnB, was partially sequenced. A cluster of six tRNA genes was found, with anticodon assignments of Asn, Thr, Gly, Arg, Pro, Ala. This cluster is closely flanked on both sides by ribosomal RNA gene sets, which were identified by sequencing upward through the 5 S rRNA gene, across a space, and into the 23 S rRNA gene, and also sequencing downward into the 16 S rRNA gene. The tRNA gene cluster appears to be organized into at least two transcriptional units separated by an attenuator region. These transcriptional units may be components of the flanking ribosomal RNA operons. The putative promoter region of the downstream 16 S rRNA is organized differently from Escherichia coli; it is smaller and seems less complex. This gene organization provides insight into possible mechanisms for coordinate and differential control of transfer and ribosomal RNA gene expression.
Anticodon↗
Molecular recognition of threonine tRNA by threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyrum pernix K1.
To investigate the recognition sites of tRNA(Thr) for threonyl-tRNA synthetase (ThrRS) from an extreme thermophilic and aerobic archaeon, Aeropyrum pernix K1, threonylation experiments using various in vitro mutant transcripts of tRNA(Thr) were examined. The results indicated that A. pernix ThrRS did recognize the first three base pairs of acceptor stem in addition to the second and the third letters of anticodon of tRNA(Thr), in spite of its N-terminal truncated unique structure. Discriminator base was not involved in recognition by A. pernix ThRS. These determinants were confirmed by the identity switching experiments from the in vitro mutants of A. pernix tRNA(Pro) and tRNA(Asn).
Archaea↗
Isolation of Chinese hamster ovary cells that overproduce asparaginyl-tRNA synthetase.
Temperature-resistant revertants, derived from the temperature-sensitive CHO asparaginyl-tRNA synthetase mutant, Asn-5, were isolated and characterized. Several lines of evidence indicate that the temperature-resistant phenotype of the revertants is due to their overproducing the same altered enzyme present in the Asn-5 parent.
Amino Acyl-tRNA Synthetases↗