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PubMed1994

Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination.

Using a genetic screen in yeast aimed at identifying cellular factors involved in initiator and elongator methionine tRNA discrimination in the translational process, we have identified a mutation that abolish the requirement for elongator methionine tRNA. The gene affected, which we call the ribosylation of the initiator tRNA gene or RIT1, encodes a 2'-O-ribosyl phosphate transferase. This enzyme modifies exclusively the initiator tRNA in position 64 using 5'-phosphoribosyl-1'-pyrophosphate as the modification donor. As the initiator tRNA participates both in the initiation and elongation of translation in a rit1 strain, we conclude that the 2'-O-ribosyl phosphate modification discriminates the initiator tRNAs from the elongator tRNAs during protein synthesis. The modification enzyme was shown to recognize the stem-loop IV region that is unique in eukaryotic cytoplasmic initiator tRNAs.

Alleles↗
PubMed1982

Selenium-containing tRNAs from Clostridium sticklandii. Cochromatography of seleno-tRNA I with L-VALYL-tRNA.

It has been previously shown that Clostridium sticklandii specifically synthesized three readily separable 75Se-labeled tRNAs, designated seleno-tRNAs I, II and III, and the partially purified seleno-tRNA II cochromatographed with L-prolyl-tRNA on DEAE-Sephadex A-50 (Chen, C.S. and Stadtman, T.C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1403-1407). In the present study a highly purified 75Se-labeled tRNA I was obtained by chromatography on benzoylated DEAE-cellulose, DEAE-Sephadex A-50 and Sepharose 4B. The 75Se-labeled tRNA I cochromatographed with an L-valine-accepting species on DEAE-Sephadex A-50 and Sepharose 4B. Addition of a 285-fold molar excess of unlabeled L-valine to the L-valine acceptor activity assay mixture markedly decreased the amount of L-[14C]valine bound to seleno-tRNA I.

Chromatography, Ion Exchange↗
PubMed1995

The presence of tRNA pseudogenes in mammalia and plants and their absence in yeast may account for different specificities of pre-tRNA processing enzymes.

Six of 13 cloned members of the human tRNA(Val) gene family code for tRNA(Val) pseudogenes, of which all but one are transcribed efficiently in HeLa cell extracts. Due to single or multiple mismatches in stem regions, the corresponding pre-tRNAs are resistant against the action of human 5'- and 3'-processing enzymes and are thus prevented from being converted to mature tRNAs. Surprisingly, all of them are accurately and efficiently processed to mature-sized tRNA in yeast nuclear extract. This is in agreement with corresponding studies of plant pre-tRNAs which are not processed in wheat germ extract but are rapidly processed in yeast extract. These observations imply that the yeast pre-tRNA 5'- and 3'-maturases do not monitor the three-dimensional structure of their substrates as stringently as mammalian and plant enzymes, possibly because tRNA pseudogenes do not occur in yeast.

Cell Extracts↗
PubMed2000

The heterotrimeric Thermus thermophilus Asp-tRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln).

Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNA(Asn) amidotransferase (Asp-AdT) capable of forming Asn-tRNA(Asn) [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832-12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln-tRNA(Gln) as well as Asn-tRNA(Asn). Thus, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated.

Amino Acid Sequence↗
PubMed2002

A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding.

The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the K(m) value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.

Adenosine Triphosphate↗
PubMed1998

Impairment of tRNA processing by point mutations in mitochondrial tRNA(Leu)(UUR) associated with mitochondrial diseases.

Several point mutations in mitochondrial tRNA genes have been linked to distinct clinical subgroups of mitochondrial diseases. A particularly large number of different mutations is found in the tRNA(Leu)(UUR) gene. We show that base substitutions at nucleotide position 3256, 3260, and 3271 of the mitochondrial genome, located in the D and anticodon stem of this tRNA, and mutation 3243 changing a base involved in a tertiary interaction, significantly impair the processing of the tRNA precursor in vitro. In correlation with other studies, our results suggest that inefficient processing of certain mutant variants of mitochondrial tRNA(Leu)(UUR) is a primary molecular impairment leading to mitochondrial dysfunction and consequently to disease.

Anticodon↗
PubMed1990

In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product.

The selD gene from Escherichia coli, whose product is involved in selenium metabolism, has been cloned and sequenced. selD codes for a protein of 347 amino acids with a calculated molecular weight of 36,687. Analysis of the selD gene product through expression of the gene in the phage T7 promoter/polymerase system confirmed the predicted molecular weight of the protein. Gene disruption experiments demonstrated that the SelD protein is required both for the incorporation of selenium into the modified nucleoside 5-methylaminomethyl-2-selenouridine of tRNA and for the biosynthesis of selenocysteine from an L-serine residue esterbonded to tRNA(Ser)(UCA). tRNA(Ser)(UCA) has been purified, aminoacylated with L-serine, and used as a substrate for the development of an in vitro system for selenocysteine biosynthesis. Efficient formation of selenocysteinyl-tRNA(Ser)(UCA) was achieved by using extracts in which both the selD and the selA gene products were overproduced. The results demonstrate that selenocysteine is synthesized from L-serine bound to tRNA(UCA) and they are in accord with SelD functioning as a donor of reduced selenium.

Amino Acid Sequence↗
PubMed2004

tRNA-triggered ATP hydrolysis and generation of membrane potential by the leishmania mitochondrial tRNA import complex.

Translocation of tRNAs across mitochondrial membranes is a receptor-mediated active transport process requiring ATP. A large tRNA import complex from the inner membrane of Leishmania mitochondria catalyzes translocation into phospholipid vesicles. In this reconstituted system, the import substrate tRNA(Tyr)(GUA) specifically stimulated hydrolysis of ATP within the vesicles, with the subsequent generation of a membrane potential by pumping out of protons, as shown by the protonophore-sensitive uptake of the potential-sensitive dye rhodamine 123. Generation of membrane potential was dependent on ATP hydrolysis, and inhibited by oligomycin, recalling the proton-translocation mechanism of the respiratory F(1)-F(0)-ATPase. For translocation of tRNA, ATP could be replaced by low pH of the medium, but proton-dependent import was resistant to oligomycin. Moreover, ATP hydrolysis, generation of membrane potential and tRNA uptake were inhibited by carboxyatractyloside, a specific inhibitor of mitochondrial ATP-ADP translocase, implying an ATP requirement within the vesicles. These observations imply a gating mechanism in which tRNA, on binding to its receptor, triggers the energetic activation of the complex, leading to the opening of import channels.

Adenosine Triphosphatases↗
PubMed1992

Pleiotropic effect of a point mutation in the yeast SUP4-o tRNA gene: in vivo pre-tRNA processing in S. cerevisiae.

The expression of mutant tyrosine-inserting ochre suppressor SUP4-o tRNA genes in vivo in S. cerevisiae was examined as a basis for further studies of tRNA transcription and processing. In vivo yeast precursor tRNAs have been identified by filter hybridization and primer extension analysis. We have previously shown that a mutant SUP4-o tRNA gene with a C52----A52 transversion at positive 52 (C52----A52(+IVS) allele) was transcribed but that the primary transcript was not processed correctly. We show here that 5' and 3' end processing as well as splicing are defective for this mutant but that the 5' end processing is restored when the intron is removed from the gene by oligonucleotide directed mutagenesis (C52----A52(-IVS) allele). Our results imply that the C52----A52 transversion by itself cannot account for the lack of susceptibility to RNase P cleavage but that the overall tertiary structure of the mutant tRNA precursor is destabilized by the intron/anticodon stem. A second consequence of the C52----A52 transversion is to prevent complete maturation of the tRNA precursor at its 3' end since intermediates containing incompletely processed 3' trailers accumulate in the yeast cells transformed with the C52----A52(-IVS) allele. A correct structure of the T stem might therefore define a structural feature required for the recognition of the 3' processing activity.

Base Sequence↗
PubMed1993

Site-directed cross-linking studies on the E. coli tRNA-ribosome complex: determination of sites labelled with an aromatic azide attached to the variable loop or aminoacyl group of tRNA.

tRNA(Phe) from E. coli, modified with the photoreactive label N-(p-azidobenzoyl)-glycine (ABG) either at the naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine (acp3U47) or the alpha-amino group of Phe-tRNA(Phe), was bound nonenzymatically to 70S ribosomes in the presence of poly (U) or short synthetic mRNA molecules prepared by T7 transcription. The noncovalent complexes were subjected to a mild ultraviolet irradiation treatment and the sites of photo-incorporation were analysed. When the photo-affinity label was attached to the aminoacyl group cross-linking was observed from both A- and P-site bound tRNA and involved exclusively the 50S subunit. In both cases the major target of cross-linking was a single site in 23S RNA, localized to position A-2439. A lower yield of cross-linking to L27 from both P- and A-sites was also observed. In contrast, cross-linking from the acp3U47 derivative was specific for P-site bound tRNA and involved mainly (but not exclusively) the 50S subunit. In this case rRNA and ribosomal protein were labelled in approximately equal yields, the sites of cross-linking involving A-2309 in 23S RNA and L33. These results are discussed in the light of our present knowledge concerning the structural arrangement of the tRNA-ribosome complex.

Affinity Labels↗
PubMed1976

On the mechanism of tRNA methylase-tRNA recognition.

In order to further elucidate the mechanism of tRNA methylase-tRNA intreaction the methylation of some individual tRNAs separately and by pairs was performed. In conditions of tRNA excess the methylation rates of positionally analogous nucleotides in tRNA molecules are not summed up when two substrates are simultaneously present in the reaction mixture. The inhibitory action of yeast tRNASer, possessing m5c in position 29, on the methylation of C29 in other individual tRNAs was shown. Yeast tRNAVal which possesses an A residue in position 27 was shown to inhibit the methylation of G27 in E. coli tRNAMet. The data obtained confirm the suggestion that tRNA methylases recognizes the tertiary structure of tRNAs. They show also that the recognition and the proper catalytic action are two autonomous processes and that the former at least in its first stage is rather unspecific.

Animals↗
PubMed1996

Automated chemical synthesis of biologically active tRNA having a sequence corresponding to Ascaris suum mitochondrial tRNA(Met) toward NMR measurements.

RNA samples corresponding to Ascaris suum mitochondrial tRNA(Met) were chemically and automatically synthesized in amounts sufficient for NMR measurement. Conventional and rapid deprotection methods gave tRNA samples with the same amino acid-accepting activity as those prepared by other method; enzymatic synthesis, and enzymatic ligation of chemically synthesized fragments. The synthetic tRNA showed the same 1H-NMR spectrum in the iminoproton region as the ligated tRNA. This rapid and reliable preparation method thus provides biologically active tRNA for NMR measurement, and further, it is applicable for synthesis of other large synthetic RNAs, by combining the site-specific isotopic labeling method.

Animals↗
PubMed2004

EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding.

Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site. Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G.

Chimera↗
PubMed1999

Mapping the active site of the Haemophilus influenzae methionyl-tRNA formyltransferase: residues important for catalysis and tRNA binding.

Formylation of the initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is an essential step in initiation of protein synthesis in eubacteria. Here, site-directed mutagenesis was used to identify active site residues of the Haemophilus influenzae MTF. Of the nine residues investigated, only Arg-41, Asn-107, His-109 and Asp-145 were important for the function of the H. influenzae MTF. Replacement of these residues with Ala resulted in a significant reduction in the efficiency of catalysis. Intrinsic fluorescence analysis indicated that this was not due to a defect in N10-formyltetrahydrofolate (fTHF) binding. The Asp-145 and Arg-41 mutations reduced the affinity of the enzyme for the initiator tRNA, whereas the Asn-107 and His-109 mutations affected catalysis but not tRNA binding. Replacement of Arg-41, His-109 and Asp-145 with functionally similar residues also affected the activity of the enzyme. The data suggest that Asn-107, His-109 and Asp-145 are catalytic residues, whereas Arg-41 is involved in tRNA recognition. In the Escherichia coli glycinamide ribonucleotide formyltransferase, which also uses fTHF as the formyl donor, Asn-106, His-108 and Asp-144 participate in the catalytic step. Together, these observations imply that this group of enzymes uses the same basic mechanism in formylating their substrates.

Amino Acid Sequence↗
PubMedDate not supplied

[The effect of modification of tRNA nucleotide-37 on the tRNA interaction with the P- and A-site of the 70S ribosome Escherichia coli].

A modified nucleotide on the 3'-side of the anticodon loop of tRNA is one of the most important structure element regulating codon-anticodone interaction on the ribosome owing to the stacking interaction with the stack of codon-anticodon bases. The presence and identity (pyrimidine, purine or modified purine) of this nucleotide has an essential influence on the energy of the stacking interaction on A- and P-sites of the ribosome. There is a significant influence of the 37-modification by itself on the P-site, whereas there is no such one on the A-site of the ribosome. Comparison of binding enthalpies of tRNA interactions on the P- or A-site of the ribosome with the binding enthalpies of the complex of two tRNAs with the complementary anticodones suggests that the ribosome by itself significantly endows in the thermodynamics of codon-anticodon complex formation. It happens by additional ribosomal interactions with the molecule of tRNA or indirectly by the stabilization of codon-anticodon conformation. In addition to the stacking, tRNA binding in the A and P sites is futher stabilized by the interactions involving some magnesium ions. The number of them involved in those interactions strongly depends on the nucleotide identity in the 37-position of tRNA anticodon loop.

Acetylation↗
PubMed1988

Biological function of modified nucleotides in tRNA molecules--synthesis and biological activity of the analogues of yeast alanyl tRNA with I34 replaced by A34 or G34.

Analogues of yeast alanyl tRNA with I34 replaced by A34 or G34 were synthesized. Synthetic analogues of yeast alanyl tRNT occupy the same position as the natural yeast alanyl tRNA on polyacrylamide gel electrophoresis, and their purity is about 95% after electrophoresis on a 10% or 20% polyacrylamide gel. The two terminal and nearest neighbour nucleotides of the analogues are all correct. The accepting activity of the synthetic analogues is similar to that of the reconstituted natural yeast alanyl tRNA. The incorporation activity of alanine into proteins of the synthetic analogues is about 30% of that of the natural of reconstituted natural yeast alanyl tRNA when I34 is replaced by A, and is 90% when I34 is replaced by G. The reason of the variation in biological function of the analogues of yeast alanyl tRNA after I34 replaced by A or G was discussed.

RNA, Fungal↗
PubMedDate not supplied

[Characteristic features of heat inactivation of leucyl-tRNA- synthetase from the rabbit liver in the presence of various tRNA conformers].

The effect of biologically inactive and active tRNA conformers on heat inactivation of leucyl-tRNA synthetase was investigated. The data presented show that inactive tRNA conformers seem to form complexes with leucyl-tRNA synthetase, but the thermal stability of the enzyme involved in the complex with inactive and active tRNA conformers is rather different.

Animals↗
PubMed1982

Isolation and characterization of an Escherichia coli mutant lacking tRNA-guanine transglycosylase. Function and biosynthesis of queuosine in tRNA.

An E. coli mutant that lacks tRNA-guanine transglycosylase was isolated by random screening from a collection of Escherichia coli mutants obtained with N-methyl-N'-nitro-N-nitrosoguanidine. The defective gene, named tgt, was mapped at about 9 min on the E. coli chromosome, and the gene order was shown to be phoB-tgt-tsx. tgt was transferred to an E. coli strain with a defined genetic background by P1 transduction to investigate its function. The mutant thus obtained lacked queuosine (2-amino-5-[3S, 4R, 5S)-4,5-dihydroxycyclopent-1-en-3-ylaminomethyl]-7-(beta-D-ribofuranosyl)-pyrro lo-[2,3-D]-pyrimidin-4-one) in tRNA, indicating that the enzyme is actually involved in the biosynthesis of queuosine in tRNA. No clear biological defect was observed in the mutant, and, in fact, it grew slightly faster than the control isogenic strain. tRNATyr lacking queuosine, isolated from the mutant, showed no significant biological difference from normal queuosine-containing tRNA in amino acid acceptor activity or amino acid transfer in a cell-free protein synthesizing system directed by synthetic polynucleotide. The only phenotypic change observed in the mutant thus far is marked reduction of viability when the cells are kept under unsuitable conditions for growth, suggesting that the presence of queuosine in tRNA is important to E. coli for survival in the natural environment.

Escherichia coli↗