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PubMed1983

Isoleucyl-tRNA synthetase from Baker's yeast. Catalytic mechanism, 2',3'-specificity and fidelity in aminoacylation of tRNAIle with isoleucine and valine investigated with initial-rate kinetics using analogs of tRNA, ATP and amino acids.

The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl-tRNA synthetase has been investigated by initial rate kinetics. For aminoacylation of tRNAIle-C-C-3'dA with isoleucine, a bi-bi uni-uni ping-pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3'dATP; for aminoacylation with valine a bi-uni uni-bi ping-pong mechanism. For isoleucylation of tRNAIle-C-C-A(3'NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni-bi uni-uni-uni ping-pong mechanism; for valylation of this tRNA a bi-bi uni-uni ping-pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle-C-C-2'dA was aminoacylated under modified conditions with isoleucine in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid-equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3'dATP and valinol. All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle-C-C-A. The Km values of the three substrates and the kcat values of the six reactions are given. For aminoacylation at the terminal 2'OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3'-OH group a difference of only 4.43 kJ and a low discrimination factor of only 6 is observed. Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2'-OH group and conclusions drawn from experiments with modified tRNAs concerning 2',3'-specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNAs. The stability of Ile-tRNAIle-C-C-2'dA and Val-tRNAIle-C-C-2'dA, the lesser stability of Val-tRNAVal-C-C-2'dA and the instability of Thr-tRNAVal-C-C-2'dA are consistent with postulations for a 'pre-transfer' proofreading step for isoleucyl-tRNA synthetase and a 'post-transfer' hydrolytic editing step for valyl-tRNA synthetase at the terminal 3'OH group of the tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Theoretical and experimental approach to recognition of amino acid by tRNA.

The C4N model concludes that the pocket on the complex of the anticodon bases and the discriminator base on a tRNA can accept the corresponding protein amino acid. A theoretical molecular computation by using the energy minimum principle is carried out to find the energy-refined C4N conformation. The results for Gln and Val suggest that not only the lock and key relationship but the induced fitting effect occurs. Some experiments to detect the interaction between the polynucleotides and the amino acid are being attempted.

Amino Acids↗
PubMed1971

Stimulation of ribonucleic acid synthesis by chloramphenicol in a rel + aminoacyl-transfer ribonucleic acid synthetase mutant of Escherichia coli.

Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel(+) strain, it cannot carry out net RNA synthesis at high temperature. A 100-mug amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 mug of CAP or 100 mug of CAP plus 50 mug of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNA(val) at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases.

Acylation↗
PubMed1970

Valine-specific tRNA-like structure in turnip yellow mosaic virus RNA.

The 3' terminal nucleotide of turnip yellow mosaic virus (TYMV) RNA (23-25 S) may be esterified with valine in the presence of ATP and an enzyme preparation from Escherichia coli. The nucleotide composition near the valine-binding site is different for TYMV RNA and tRNA(Val) from cabbage, as shown by comparison of the valine adducts of nucleotides labeled with radioactive valine in T(1) RNase digests. Consequently, host tRNA(Val) is not involved in the observed charging of TYMV RNA with valine. The TYMV RNA appears to have a tRNA-like structure, at or near its 3' end, that is recognized by three different enzymes which specifically catalyze reactions involving tRNA.

Binding Sites↗
PubMed1992

The first human genes for tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU) and more tRNA(Val) pseudogenes: expression and pre-tRNA maturation in HeLa cell-free extracts.

A functional tRNA(Val) gene, which codes for the major tRNA(ValIAC) isoacceptor species, and three new tRNA(Val) pseudogenes have been isolated from human genomic DNA. Two tRNA(Val) pseudogenes and a tRNA(Val) variant gene were found to be associated with tRNA genes encoding tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU), respectively, on distinct DNA fragments. All tRNA genes, including the pseudogenes, are actively transcribed in HeLa nuclear extract. Pre-tRNAs of tRNA(Val), tRNA(Arg), tRNA(Thr), and tRNA(Gly) genes are correctly processed to mature-sized tRNAs, whereas the three tRNA(Val) pseudogenes yield stable pre-tRNAs in vitro. These findings reveal that, together with the three known pseudogenes, half of the members of the human tRNA(Val) gene family are pseudogenes, all of which are active in homologous nuclear extracts in vitro and presumably also in vivo.

Base Sequence↗
PubMed1992

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↗
PubMed1984

Halobacterium volcanii tRNAs. Identification of 41 tRNAs covering all amino acids, and the sequences of 33 class I tRNAs.

Transfer RNAs of Halobacterium volcanii, an archaebacterium, were separated by two-dimensional gel electrophoresis and sequenced by a combination of methods. A total of 41 tRNAs, at least one for each amino acid, were identified. These are five tRNAs for Leu, four for Gly, three each for Ala, Arg, Pro, and Ser, two each for Glu, Ile, Lys, Met (initiator and noninitiator), Thr, and Val, and one each for the remaining eight amino acids. As in eucaryotes, only Leu and Ser tRNAs are class II (large extra arm). The sequences of the 33 class I tRNAs, for the remaining 18 amino acids, are presented here. These cover at least 44 codons out of a possible 49 codons for the 18 amino acids. Although these archaebacterial tRNAs follow general tRNA patterns, they are in detail distinct from both eucaryotic and eubacterial tRNAs. Moreover, the initiator tRNA is unique in having a 5'-triphosphorylated end.

Amino Acids↗
PubMed1991

Purification and properties of a high-molecular-mass complex between Val-tRNA synthetase and the heavy form of elongation factor 1 from mammalian cells.

In extracts of various mammalian tissues obtained in the presence of protease inhibitors Val-tRNA synthetase exists exclusively as a complex with a molecular mass of about 800 kDa. This complex was purified by gel filtration and two HPLC steps and contained five different polypeptides with molecular masses of 140, 50, 50, 40 and 30 kDa. The complex seems to have no tissue or species specificity, because preparations with identical polypeptide composition were obtained by the same method from rabbit liver and reticulocytes, and rat and beef liver. Four low-molecular-mass polypeptides were identified by two-dimensional electrophoresis as subunits of the heavy form of elongation factor 1 (EF-1H). The complex possesses the activity of EF-1 in the poly(U)-directed translation system, indicating that EF-1H is an integral part of the complex. Gel filtration of the tissue extracts reveals three different peaks of EF-1 activity, corresponding to EF-1 alpha, EF-1H and the high-molecular-mass complex of Val-tRNA synthetase and EF-1H. All activity of Val-tRNA synthetase and about 25% of EF-1 activity are associated with the complex. Different forms of EF-1 revealed no significant differences in the nucleotide-binding properties, but the complex of Val-tRNA synthetase with EF-1H was 10 times more active in the poly(U)-directed binding of Phe-tRNAPhe to ribosomes than EF-1H. These results strongly suggest that the complex of Val-tRNA synthetase with EF-1H is a novel functionally active individual form of EF-1.

Animals↗
PubMed1993

Nucleotide sequence of a Trichophyton mentagrophytes HindIII mitochondrial DNA fragment containing a tRNA gene cluster.

A 0.85-kb HindIII mitochondrial DNA fragment of the dermatophytic fungus Trichophyton mentagrophytes has been sequenced. The fragment contains eight complete genes which corresponds to a tRNA gene cluster. From 5' to 3', the sequenced genes code for tRNA(thr), tRNA(glu), tRNA(val), tRNA(met1), tRNA(met3), tRNA(leu), tRNA(ala), and tRNA(phe). This tRNA gene cluster is located downstream of the larger ribosomal RNA gene. The particularities of the sequenced genes and their comparison with other fungal tRNA mitochondrial genes are reported.

Base Sequence↗
PubMed1984

Does codon composition influence ribosome function?

Escherichia coli ribosomes pre-initiated with N-acetyl-Val-tRNAVal elongate strictly alternating poly(U-G) at a rate between eight and 12 peptide bonds per second per ribosome in vitro. Comparisons with poly(U)-primed poly(Phe) synthesis show that these systems function with the same rates which are close to those of protein synthesis in vivo. This indicates that, at least in vitro, codon composition has no marked influence on the speed of elongation when the concentration of ternary complex is saturating. Furthermore, the missense frequencies for the two polymers are within the same range: the missense substitution of Trp for Cys is 10(-4) and that of Met for Val is 10(-3) in the poly(U-G)-primed system. These data argue against models that explain the codon preference of certain gene families by postulating effects of high or low GC content of codons on the performance characteristics of ribosomes.

Codon↗
PubMed1987

Chromosomal mapping of tRNA genes from Dictyostelium discoideum.

Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA Val(GUU) and a tRNA Val(GUA) gene. The method used involved a combination of classical D. discoideum parasexual genetics and molecular genetics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VII (A, H). Six tRNA Val(GUA) genes from the NC4 isolate were mapped: one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).

Chromosome Mapping↗
PubMed1981

The nucleotide sequence of the large ribosomal RNA gene and the adjacent tRNA genes from rat mitochondria.

We have sequenced the Eco R(1) fragment D from rat mitochondrial DNA. It contains one third of the tRNA (Val) gene (the remaining part has been sequenced from the 3' end of the Eco R(1) fragment A) the complete gene for the large mt 16S rRNA, the tRNA (Leu) gene and the 5' end of an unidentified reading frame. The mt gene for the large rRNA from rat has been aligned with the homologous genes from mouse and human using graphic computer programs. Hypervariable regions at the center of the molecule and highly conserved regions toward the 3' end have been detected. The mt gene for tRNA Leu is of the conventional type and its primary structure is highly conserved among mammals. The mt gene for tRNA(Val) shows characteristics similar to those of other mt tRNA genes but the degree of homology is lower. Comparative studies confirm that AGA and AGG are read as stop codons in mammalian mitochondria.

Animals↗
PubMed1986

Interaction of turnip yellow mosaic virus Val-RNA with eukaryotic elongation factor EF-1 [alpha]. Search for a function.

The 3'-terminal tRNA-like structure in turnip yellow mosaic virus (TYMV) RNA can be adenylated by tRNA nucleotidyltransferase and subsequently aminoacylated by valyl-tRNA synthetase. Here we present evidence that TYMV Val-RNA can form a stable complex with eukaryotic wheat germ elongation factor EF-1alpha and GTP: the Val-RNA is protected by EF-1alpha.. GTP against digestion by RNase A. By affinity chromatography of TYMV Val-RNA fragments on immobilized EF-1alpha . GTP, it has been established that the valylated aminoacyl RNA domain, which in TYMV RNA is formed by the 3' half of the tRNA-like region, is sufficient for complex formation with EF-1alpha . GTP. The aminoacyl RNA domain is equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T stem and loop. In line with these results, the aminoacyl RNA domain in TYMV Val-RNA complexed to EF-1 alpha . GTP is resistant to digestion by RNase A. It is also shown that the TYMV RNA replicase (RNA-dependent RNA polymerase) isolated from TYMV-infected Chinese cabbage leaves does not contain tRNA nucleotidyltransferase, valyl-tRNA synthetase or EF-1alpha. This suggests that interaction of TYMV RNA with EF-1alpha is not mandatory for replicase activity.

Base Sequence↗
PubMed1977

Study of yeast mitochondrial tRNAs by two-dimensional polyacrylamide gel electrophoresis: characterization of isoaccepting species and search for imported cytoplasmic tRNAs.

By two-dimensional polyacrylamide gel electrophoresis, yeast mitochondrial tRNA is fractionated into 27 major species. All but 6 of them migrate distinctly from cytoplasmic tRNAs. Migration of mitochondrial DNA-coded mitochondrial tRNAs shows the occurence of only one cytoplasmic tRNA in mitochondria. Several mitochondrial tRNA spots are identified on the electrophoregrams, some of them show isoaccepting species (Val, Ser, Met, Leu). It is suggested that there are sufficient mitochondrial tRNA genes on yeast mitochondrial DNA to allow mitochondrial protein biosynthesis by the mitochondrial tRNAs alone. Guanosine + Cytidine content and rate base composition are reported for some individual species. Mitochondrial tRNAPhe lacks Ribothymidine.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Transfer ribonucleic acid deprived of the C-C-A 3'-extremity can interact with elongation factor Tu.

In this work we describe that uncharged tRNAs or modified tRNAs lacking all or part of the C-C-A end (i.e., tRNA minus pCpCpA, tRNA minus pA, and tRNA minus A) can still influence the GTPase activity of the elongation factor Tu (EF-Tu), thus showing that, besides the aminoacylated 3'-end, other regions of the aa-tRNA interact with EF-Tu. The existence of an interaction between EF-Tu and truncated tRNAs was also confirmed by examining the dissociation of the EF-Tu-GTP complex: the rate of this reaction is decreased upon addition of tRNAVal1 minus pCpCpA. The effect on the EF-Tu GTPase activity of tRNAs deprived of the C-C-A 3'-end is still evident in the presence of C-C-A-aa. The stimulatory pattern obtained with C-C-A-Val at 5 mM MgCl2 is decreased upon addition of tRNAVal1 minus pCpCpA, tRNAVal1 minus pA, or tRNAVal1 minus A. This shows that the effect of the aminoacylated C-C-A 3'-end can be influenced via EF-Tu by the remaining regions of the tRNA, after cleavage of a bond in the 3'-extremity. However, also with an excess of tRNAVal1 minus pCpCpA over C-C-A-Val, no "aa-tRNA-like" effect, i.e., no inhibition of the EF-Tu GTPase, was obtained, suggesting that, upon binding with EF-Tu, a specific conformational change in the aa-tRNA molecule also takes place, regulating the expression of the GTPase activity. Our results unequivocally show that different regions of the aa-tRNA are needed for a coordinated interaction with EF-Tu.

Base Sequence↗
PubMed1989

Isoleucyl-tRNA synthetase from baker's yeast. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA; role of terminal hydroxyl groups aminoacylation of tRNA(Ile)-C-C-A.

Specificity with regard to amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA by isoleucyl-tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2 = 180-1700), indicating that at same amino acid concentrations isoleucine is 180-1700 times more attached to tRNA(Ile)-C-C-3'dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2 = 10,000-30,000). D2 values of the other amino acids are in the range of 2000-10,000. Recognition of most amino acids is achieved in a four-step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof-reading steps occur on the pre- and the post-transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post-transfer proof-reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post-transfer proof-reading step are lacking. The role of the terminal hydroxyl groups of the tRNA for post-transfer proof-reading is assigned to a simple neighbouring group effect. No preference for the 2' or 3' position in proof-reading can be postulated.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Codon specificity of UGA suppressor tRNATrp from Escherichia coli.

A synthetic polyribonucleotide, poly(U5,G), was used to study the codon specificity of wild-type and UGA suppressor tRNATrp from Escherichia coli. Phe (UUU) incorporation directed by this synthetic messenger is reduced somewhat by omission from the incubation mixtures of Val (GUU), Leu (UUG), or Cys (UGU). In contrast, omission of Cys stimulates Trp incorporation, and this effect is much more pronounced with the UGA suppressor tRNATrp than with wild-type tRNA. The apparent replacement of Cys by Trp is specific, because the omission of Val or Leu slightly inhibits Trp incorporation. These data suggest that the UGA suppressor tRNATrp can translate codons of the form UGN (N is any ribonucleotide). In other words, the suppressor tRNATrp translates codons that properly match two out of the three anticodon nucleotides.

Base Sequence↗