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At least 19 recordsLinked to original sources
PubMed1993

Two Streptomyces lividans 66 transfer RNA genes with anticodons corresponding to serine (AGC) and arginine (CGU) codons.

From the nucleotide sequence of a 2110-bp Streptomyces lividans 66 DNA fragment two transfer RNA genes were identified: tRNA(Ser) (AGC) and tRNA(Arg) (CGU). These tRNA genes are transcribed from the same DNA strand and both are preceded by a putative promoter structure. They are separated by an intergenic region of 191 bp. Like most Streptomyces tRNA genes described so far, they do not encode the 3' terminal CCA of mature tRNAs. Both genes are followed by extensive inverted repeats, which could serve as transcriptional terminator signals. Remarkably, these hairpin structures share an identical 9 base pair stretch (5'-GAAGCCCCG-3'). Furthermore, the tRNA(Arg) region is followed by two potential open reading frames, which are encoded on complementary strands and have 3' overlapping ends. The gene for tRNA(Arg) (CGU) is the first Streptomyces tRNA gene described so far which encodes the translation of a codon with uridine at the third (wobble) position.

Anticodon↗
PubMed1992

Self-splicing introns in tRNA genes of widely divergent bacteria.

The organization of eukaryotic genes into exons separated by introns has been considered as a primordial arrangement but because it does not exist in eubacterial genomes it may be that introns are relatively recent acquisitions. A self-splicing group I intron has been found in cyanobacteria at the same position of the same gene (that encoding leucyl transfer RNA, UAA anticodon) as a similar group I intron of chloroplasts, which indicates that this intron predates the invasion of eukaryotic cells by cyanobacterial endosymbionts. But it is not clear from this isolated example whether introns are more generally present in different genes or in more diverse branches of the eubacteria. Many mitochondria have intron-rich genomes and were probably derived from the alpha subgroup of the purple bacteria (or Proteobacteria), so ancient introns might also have been retained in these bacteria. We describe here the discovery of two small (237 and 205 nucleotides) self-splicing group I introns in members of two proteobacterial subgroups, Agrobacterium tumefaciens (alpha) and Azoarcus sp. (beta). The introns are inserted in genes for tRNA(Arg) and tRNA(Ile), respectively, after the third anticodon nucleotide. Their occurrence in different genes of phylogenetically diverse bacteria indicates that group I introns have a widespread distribution among eubacteria.

Base Sequence↗
PubMed1988

A yeast tRNA(Arg) gene can act as promoter for a 5' flank deficient, non-transcribable tRNA(SUP)6 gene to produce biologically active suppressor tRNA.

In S. cerevisiae most tRNA genes are located and expressed as single entities. The tDNA(Arg)-tDNA(Asp) pair, however, is transcribed into a dimeric precursor before being processed into two mature tRNA species. The second gene of this pair, tDNA(Asp), is totally dependent on the first gene, tDNA(Arg), and its promoter components, for homologous in vitro transcription. The second gene in the pair is now replaced by the ochre suppressor tDNA(SUP)6-o, which, by itself, cannot be transcribed because of a nonfunctional 5' flanking region. The tDNA(Arg)-tDNA(SUP)6-o was transcribed into a dimeric precursor which was processed to mature tRNA molecules as judged in vitro by electrophoretic separation, and in vivo by their ability to suppress ochre but not amber yeast mutations. Mutations in the internal promoter of the first gene decreased transcription, both in vitro and in vivo, of the second-tRNA(SUP)6-o-gene. Thus tDNA(Arg) with its 5' flanking region can act as an external promoter for other RNA polymerase III-read genes that are by themselves inactive due to impaired promoter/modulator regions.

Base Sequence↗
PubMed1992

In vitro study of E.coli tRNA(Arg) and tRNA(Lys) identity elements.

Various tRNA transcripts were constructed to study the identity elements of E.coli tRNA(Arg) and tRNA(Lys). Exchange of the anticodon of the major tRNA(Arg) from ACG to either CCG or CCU did not result in a significant loss of arginine acceptor activity, whereas not only that to UUU but also that to ACA or ACC decreased the activity. Base substitutions and deletion at A20 also impaired the arginine charging activity by over 50-fold. Arginine charging activity was introduced by either substitution of the anticodon from UAC to ACG in tRNA(Val) or from UUU to UCU in tRNA(Lys). Only a single base substitution at the third position of tRNA(Trp) anticodon (CCA) from A to G also gave rise to arginine charging activity, which was elevated to a comparable level to that of the tRNA(Arg) transcript by an additional A20 insertion. Base substitutions of the major tRNA(Arg) at the discriminator position into pyrimidines led to a decrease by factors of three to four. These data show that the third letter of the anticodon G36 or U36 besides the second letter C35 and the A20 in the variable pocket is responsible for the arginine acceptor identity, to which the discriminator base A73 or G73 contributes in an auxiliary fashion. In contrast to the arginine system, the transcript with the wild-type tRNA(Lys) sequence showed only 140-fold lower lysine charging activity than the native tRNA(Lys), suggesting the involvement of base modifications in recognition. Replacement of the anticodon UUU with not only UCU and UAC but also UUA and UUC seriously affected the lysine acceptor activity, and those with GUU and UUG also decreased by factors of 17 and 5, respectively. Introduction of UUU into the anticodons conferred lysine charging activity upon both tRNA(Val) and tRNA(Arg). Substitution of the discriminator base A73 by any of the other bases decreased the lysine acceptor activity by a factor of ten. These results indicate the involvements of all the three bases of the anticodon and A at the discriminator position in lysine specific aminoacylation.

Anticodon↗
PubMed1991

Chromosomal assignment of a large tRNA gene cluster (tRNA(Leu), tRNA(Gln), tRNA(Lys), tRNA(Arg), tRNA(Gly)) to 17p13.1.

A cluster of tRNA genes (tRNA(UAGLeu), tRNA(CUGGln), tRNA(UUULys), tRNA(UCUArg)) and an adjacent tRNA(GCCGly) have been assigned to human chromosome 17p12-p13.1 by in situ hybridization using a 4.2 kb human DNA fragment for tRNA(Leu), tRNA(Gln), tRNA(Lys), tRNA(Arg), and, for tRNA(Gly), 1.3 kb and 0.58 kb human DNA fragments containing these genes as probes. This localization was confirmed and refined to 17p13.100-p13.105 using a somatic cell hybrid mapping panel. Preliminary experiments with the biotinylated tRNA Leu, Gln, Lys, Arg probe and metaphase spreads from other great apes suggest the presence of a hybridization site on the long arm of gorilla (Gorilla gorilla) chromosome 19 and the short arm of orangutan (Pongo pygmaeus) chromosome 19 providing further support for homology between HSA17, GGO19 and PPY19.

Animals↗
PubMed1994

Factor for inversion stimulation-dependent growth rate regulation of individual tRNA species in Escherichia coli.

We have studied the involvement of the factor for inversion stimulation (FIS) in the growth rate-dependent expression of the arginine, leucine, and methionine acceptor tRNA species. The concentration of individual tRNA species relative to 16 S rRNA was determined by blot hybridization using RNA preparations from bacteria with the fis gene deleted and from isogenic wild type bacteria. The RNA preparations were obtained from bacteria growing under steady state conditions in different media. The levels of tRNA(1Leu), tRNA(2Arg), tRNA(4Arg), and tRNA(5Arg decreased in the fis bacteria, relative to the wild type. The difference in levels increased with increasing growth rate. Surprisingly, tRNA(3Leu), tRNA(rMet), and tRNA(eMet) showed the opposite response, with an increase of the tRNA/16 S ratio in the fis bacteria. The tRNA(2Leu, tRNA(4Leu), tRNA(5Leu), and tRNA(3 Arg) had unaffected tRNA/16 S ratios in fis cells. We conclude that FIS, directly or indirectly, is involved in growth rate regulation of some tRNA species and that it affects the composition of the cellular tRNA pool.

Base Sequence↗
PubMed1996

Arginine aminoacylation identity is context-dependent and ensured by alternate recognition sets in the anticodon loop of accepting tRNA transcripts.

Yeast arginyl-tRNA synthetase recognizes the non-modified wild-type transcripts derived from both yeast tRNA(Arg) and tRNA(Asp) with equal efficiency. It discriminates its cognate natural substrate, tRNA(Arg), from non-cognate tRNA(Asp) by a negative discrimination mechanism whereby a single methyl group acts as an anti-determinant. Considering these facts, recognition elements responsible for specific arginylation in yeast have been searched by studying the in vitro arginylation properties of a series of transcripts derived from yeast tRNA(Asp), considered as an arginine isoacceptor tRNA. In parallel, experiments on similar tRNA(Arg) transcripts were performed. Unexpectedly, in the tRNA(Arg) context, arginylation is basically linked to the presence of residue C35, whereas in the tRNA(Asp) context, it is deeply related to that of C36 and G37 but is insensitive to the nucleotide at position 35. Each of these nucleotides present in one host, is absent in the other host tRNA. Thus, arginine identity is dependent on two different specific recognition sets according to the tRNA framework investigated.

Anticodon↗
PubMed1990

An archaebacterial cell-free transcription system. The expression of tRNA genes from Methanococcus vannielii is mediated by a transcription factor.

Our understanding of the mechanism of RNA biosynthesis in archaebacteria is limited, due in part to the inability of purified RNA polymerases to transcribe purified genes accurately in vitro. In the present study, we show that cell extracts of Methanococcus vannielii and Methanococcus thermolithotrophicus purified by gradient centrifugation synthesize a distinct transcript from templates harboring a cloned homologous tRNA(Val) and tRNA(Arg) gene. The in vitro transcripts initiate with GTP at the same sites as in Methanococcus cells. About 60% of the sequence of the in vitro RNA products was analyzed by dideoxyterminated primer extension and found to be identical with that of the precursors of tRNA(Val) and tRNA(Arg). This finding indicates that this RNA polymerase fraction both initiates and terminates transcription faithfully in vitro. After purification of a cell-free extract (S-100) of M. thermolithotrophicus by phosphocellulose chromatography, the endogenous RNA polymerase has lost its ability to transcribe the tRNA(Val) gene accurately. The activity directing specific expression of this template was reconstituted by the addition of a protein-fraction devoid of RNA polymerase activity. Thus, a transcription factor appears to be required for accurate cell-free expression of tRNA genes from M. vannielii.

Base Sequence↗
PubMed1998

The RNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA.

Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNA(Arg) and C36 and G37 in tRNA(Asp) (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069-5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNA(Arg) and tRNA(Asp), the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899-4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different k(cat) and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system.

Anticodon↗
PubMed1990

A set of tRNAs that lack either the T psi C arm or the dihydrouridine arm: towards a minimal tRNA adaptor.

The mitochondrial DNA (mtDNA) molecules of the nematode worms, Caenorhabditis elegans and Ascaris suum contain 22 putative genes for non-standard forms of tRNAs. The inferred transcripts can be folded into 20 separate structures each resembling a tRNA whose T psi C arm and variable loop are replaced with a simple loop of 6-12 nucleotides. In two further structures [that resemble tRNAs for ser(UCN) and ser(AGN)], the dihydrouridine arm is replaced by a loop of 5-8 nucleotides. By hybridizing mt-tRNA gene-specific oligonucleotide probes to nematode RNAs, we have obtained evidence for transcription of at least nine C.elegans and three A.suum mt-tRNA genes. Each transcript (tRNA) is the exact size predicted from the respective DNA sequence, to which three nucleotides, presumably CCA, have been added following transcription. An exception was C.elegans mt-tRNAasn, most molecules of which had one nucleotide (plus CCA) more than predicted from the gene. The data presented strongly support the conclusion that the functional mt-tRNAs of nematode worms are direct transcripts (with only CCA addition) of the structurally unusual mt-tRNA genes. There is no evidence of trans-splicing or RNA editing to add the sequences missing from these nonstandard tRNAs. We presume, therefore, that the non-standard forms are active in mitochondrial protein synthesis.

Animals↗
PubMed1999

Cloning, sequencing, and characterization of ribosomal protein and RNA polymerase genes from the region analogous to the alpha-operon of escherichia coli in halophilic archaea, halobacterium halobium.

A determination was made of the nucleotide sequence of the 3215-bp region of a ribosomal protein gene cluster (HS13, HS4, HS11, and HeL18), RNA polymerase (RNA poly D), and tRNA genes (tRNAser and tRNAarg) of halophilic Archaea Halobacterium halobium, which is analogous to the alpha-operon of Escherichia coli (tRNAser-HS13-HS4-HS11-RNA poly D-tRNAarg-HeL18). The seven-gene string was preceded by a pseudoknot-like structure similar to the proposed S4 ribosomal protein binding site of the alpha-operon mRNA leader in E. coli. Using an inducible expression system H. halobium HS4 was produced in large amounts in E. coli, and immunoblot analysis showed the S4 to constitute a 21-kDa polypeptide component of the ribosome. Analysis of the deduced amino acids sequence revealed that the HS13, HS4, and HS11 sequences including the RNA polymerase subunit are more similar to their eukaryotic than to their bacterial counterparts. HeL18, located downstream of the gene cluster analogous to the E. coli alpha-operon (S13-S11-S4-RNA poly D-L17), was similar to both the eukaryotic (eL18) and eubacterial ribosomal protein L15 located in the spc-operon, but not to L17 positioned as the terminal gene of the bacterial alpha-operon.

Amino Acid Sequence↗
PubMed1996

A cluster of transfer RNA genes (TRM1, TRR3, and TRAN) on the short arm of human chromosome 6.

We have isolated two lambda clones that contain three transfer RNA (tRNA) genes (TRM1, TRR3, and TRAN). Both clones map to the same region (6p21.2-p22.3) of the short arm of chromosome 6. One clone contains a methionine tRNA gene and also an arginine tRNA gene, the first such human gene to be described. The other clone contains an alanine tRNA gene, again the first such human gene to be reported, and it differs from the species of human alanine tRNA transcripts sequenced to date. These clones have been used to investigate the structure of this tRNA gene cluster. The results of both conventional and pulsed-field gel analysis suggest that the alanine tRNA gene is a member of a low-copy repeat series at this location. The other clone is not located within this domain and appears to be a unique segment of DNA. Nevertheless, we also show that at least half of the methionine tRNA genes are located on the short arm of this chromosome, and if these are also located at 6p21.2-p22.3, this would constitute another major tRNA locus in human.

Bacteriophage lambda↗
PubMed1997

Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner.

In Salmonella typhimurium seven tRNA species specific for leucine, proline and arginine have 1-methylguanosine (m1G) next to and 3' of the anticodon (position 37 of tRNA), five tRNA species specific for phenylalanine, serine, tyrosine, cysteine and tryptophan have 2-methylthio-N-6-(cis-hydroxy)isopentenyladenosine (ms2io6A) in the same position of the tRNA, and four tRNA species, specific for leucine and proline, have pseudouridine (Psi) as the last 3' nucleotide in the anticodon loop (position 38) or in the anticodon stem (positions 39 and 40). Mutants deficient in the synthesis of these modified nucleosides have been used to study their role in the first step of translation elongation, i.e. the aa-tRNA selection step in which the ternary complex (EF-Tu-GTP-aa-tRNA) binds at the cognate codon in the A-site on the mRNA programmed ribosome. We have found that the Psi present in the anticodon loop (position 38) stimulates the selection of tRNA specific for leucine whereas Psi in the anticodon stem did not affect the selection of tRNA specific for proline. The m1G37 strongly stimulates the rate of selection of the three tRNA species specific for proline and one tRNA species specific for arginine but has only minor or no effect on the selection of the three tRNA species specific for leucine. Likewise, the ms2io6A, present in the same position as m1G37 but in another subset of tRNA species, stimulates the selection of tRNA specific for tyrosine, stimulates to some extent also tRNA species specific for cysteine and tryptophan, but has no influence on the rate of selection of tRNA specific for phenylalanine. We conclude that function of m1G and ms2io6A present next to and 3' of the anticodon influences the in vivo aa-tRNA selection in a tRNA-dependent manner.

Anticodon↗
PubMed1985

Location and nucleotide sequence of the genes for tobacco chloroplast tRNAArg (ACG) and tRNALeu(UAG).

The location and nucleotide sequence of the genes and flanking regions for tRNAArg(ACG) and tRNALeu(UAG) on tobacco chloroplast DNA have been determined. The gene arrangement is 5S rRNA-260 bp-tRNAArg-581 bp-tRNAAsn-5.2 kbp-tRNALeu. The tRNAArg and tRNALeu genes are expressed in the chloroplasts. The opposite strand of the tRNAArg gene contains a tRNAArg-like sequence. The tRNAArg, tRNAAsn and tRNALeu coding regions are contained in open reading frames.

Base Sequence↗
PubMed1994

Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis.

Nonnatural amino acids with photofunctional groups were incorporated site-specifically into a polypeptide by using in vitro protein synthesizing system. The nonnatural amino acids were attached to tRNA(CCU) through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon. L-p-Phenylazophenylalanine, L-2-anthrylalanine, L-1-naphthylalanine, L-2-naphthylalanine and L-p-biphenylalanine were successfully incorporated into a polypeptide, but l-1-pyrenylalanine was not. The polypeptides containing the nonnatural amino acids showed photofunctionalities.

Acylation↗
PubMed1996

Localization and expression of the closely linked cyanelle genes for RNase P RNA and two transfer RNAs.

The genomic region encoding the RNA subunit of the cyanelle RNase P has been characterized. rnpB, which has no homologue in chloroplasts, is flanked by two tRNA genes on the complementary DNA strand. Transcriptional control elements of all three genes have been experimentally determined. Comparison of the sequenced region with the corresponding loci of chloroplast genomes from vascular plants suggests that major inversions may have led to a possible loss or severe truncation of the RNase P RNA coding region during the course of plastid evolution.

Base Sequence↗
PubMed1992

Seven, eight and nine-membered anticodon loop mutants of tRNA(2Arg) which cause +1 frameshifting. Tolerance of DHU arm and other secondary mutations.

The mutant tRNA(2Arg) encoded by the genetically-selected frameshift suppressor, sufT621, inserts arginine and causes a +1 reading-frame shift at the proline codon, CCG(U). There is an extra base, G36.1, in argV beta, one of the four identical genes for tRNA(2Arg) in the position between bases 36 and 37, corresponding to the 3' side of the anticodon. The new four-base anticodon, predicted from DNA sequencing to be 3' GGCA 5', is complementary to the four-base codon CCGU. Quadruplet translocation promoted by mutant argV does not require perfect complementarity between the codon and the anticodon since synthetic genes encoding derivatives of tRNA(2Arg) and tRNA(1Pro), with four-base anticodons complementary to three out of the four bases of CCGU, were also shown to be capable of frameshifting. Two other mutants of argV, inferred to have normal-size, seven-base anticodon loops, were also found to be capable of four-base-decoding demonstrating that quadruplet translocation promoted by mutant argV does not require an enlarged anticodon loop. Other alleles of argV, predicted to have nine bases in the anticodon loop, were also found to cause frameshifting. The DNA sequence of two of these showed in addition, either a deletion of G24, or a ten-base duplication in the region corresponding to the TFC arm. A general finding is that mutations in the DHU arm of tRNA(2Arg) are compatible with, and in one case necessary for, frameshifting.

Amino Acid Sequence↗