Specific conversion of s4 U to U in Escherichia coli tRNA by iodate oxidation.

The minor nucleoside 4-thiouridine in Escherichia coli tRNA is transformed selectively to uridine by iodate oxidation at acidic pH. The four major nucleotides were found to be inert under these conditions. The iodate oxidation appears to be more specific than the previous conversion methods reported, and has the advantage that it does not affect the chargeability of most tRNA.     

Arginyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2)

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10,000 and 60,000. In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role. Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed.     

The nucleotide sequence of a methionine tRNA which functions in protein elongation in mouse myeloma cells.

The major form of methionine tRNA operational in the elongation of protein synthesis in mouse myeloma cells was purufied from these cells after they had been cultured in the presence of [32P]-phosphate. This [32P]tRNA4-Met species was then digested with T1 RNase or pancreatic RNase so as to obtain both complete and partial RNase digestion products. The nucleotide sequences of these fragments were analysed to enable the derivation of the complete primary structure of this tRNA. tRNA4-Met of mouse myeloma cells is 76 nucleotides in length and contains 15 modified nucleotides. It is the only tRNA yet sequenced which has been found to possess the minor nucleoside 2-methylguanosine (m2G) within the amino acid (a) stem, and also to have an anticodon (c) stem of only 4 and not 5 base-pairs. The loop IV sequence of eukaryotic initiator methionine tRNA (tRNAf-Met) species, -A-U-C-G-m1A-A-A-, IS NOT FOUND IN TRNA4-Met and is therefore absent from at least one of the methionine tRNAs functioning in polypeptide elongation in mammalian cells. This is consistent with the suggested importance of this loop structure in the initiator function of tRNAf-Met in eukaryotic organisms. Three distinct regions of the tRNA cloverleaf, the (b) stem, the anticodon loop (loop II), and loop III, are substantially conserved in structure between tRNAf-Met and tRNA4-Met of mouse myeloma cells. These regions of the structures of mammalian methionine tRNAs probably do not determine whether a certain tRNA-Met will function in the initiation or elongation of protein synthesis, although they might be important in tRNA-Met recognition if the different cytoplasmic tRNA-Met species of mammalian cells are aminoacylated by a single activating enzyme.     

tRNA leucine identity and recognition sets

Transfer RNAs (tRNAs) are grouped into two classes based on the structure of their variable loop. In Escherichia coli, tRNAs from three isoaccepting groups are classified as type II. Leucine tRNAs comprise one such group. We used both in vivo and in vitro approaches to determine the nucleotides that are required for tRNA(Leu) function. In addition, to investigate the role of the tRNA fold, we compared the in vivo and in vitro characteristics of type I tRNA(Leu) variants with their type II counterparts.A minimum of six conserved tRNA(Leu) nucleotides were required to change the amino acid identity and recognition of a type II tRNA(Ser) amber suppressor from a serine to a leucine residue. Five of these nucleotides affect tRNA tertiary structure; the G15-C48 tertiary "Levitt base-pair" in tRNA(Ser) was changed to A15-U48; the number of nucleotides in the alpha and beta regions of the D-loop was changed to achieve the positioning of G18 and G19 that is found in all tRNA(Leu); a base was inserted at position 47n between the base-paired extra stem and the T-stem; in addition the G73 "discriminator" base of tRNA(Ser) was changed to A73. This minimally altered tRNA(Ser) exclusively inserted leucine residues and was an excellent in vitro substrate for LeuRS. In a parallel experiment, nucleotide substitutions were made in a glutamine-inserting type I tRNA (RNA(SerDelta); an amber suppressor in which the tRNA(Ser) type II extra-stem-loop is replaced by a consensus type I loop). This "type I" swap experiment was successful both in vivo and in vitro but required more nucleotide substitutions than did the type II swap. The type I and II swaps revealed differences in the contributions of the tRNA(Leu) acceptor stem base-pairs to tRNA(Leu) function: in the type I, but not the type II fold, leucine specificity was contingent on the presence of the tRNA(Leu) acceptor stem sequence. The type I and II tRNAs used in this study differed only in the sequence and structure of the variable loop. By altering this loop, and thereby possibly introducing subtle changes into the overall tRNA fold, it became possible to detect otherwise cryptic contributions of the acceptor stem sequence to recognition by LeuRS. Possible reasons for this effect are discussed.     

Synthesis of RNA containing O-beta-D-ribofuranosyl-(1'-2')-adenosine-5'-phosphate and 1-methyladenosine, minor components of tRNA

tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is well-known based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-beta-D-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5'-O-phosphorylated 2'-O-(beta-D-ribofuranosyl)-beta-D-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA(Met) will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.     

5' terminal nucleotide sequences of the messenger RNA's of Dictyostelium discoideum.

As in the mRNA from all other eucaryotic cells examined, the 5' nucleotide in messenger RNA from Dictyostelium discoideum is linked by a 5'-5' triphosphate bridge to the unusual nucleoside 7-methyl guanosine. In mammalian cellular mRNA, the 5' terminal sequences have the general formula m7GpppXmpYp(m), where X and Y can be either purine or pyrimidine nucleotides and Y, as well as X, may contain a 2'0-methylated ribose. Although at least 32 5' terminal sequences are possible in cellular mRNA, only four are present in Dictyostelum mRNA. They are (I) m7GppppAp (65%); (II) m7GpppGp (10%); (III) m7GpppAmpAp (10%); (IV m7GpppAp (65%); (II) m7gpppGp (10%); (III) m7GpppAmpAp (10%); (IV) m7GpppAmpUp (10%). Sequences I and II are simpler than those previously reported for mammalian cells because they lack 2'0-methylated nucleosides. Another difference is that in all Dictyostelium mRNAs. the nucleoside X is a purine. The nucleoside 6-methyl adenosine which is found internal to the 5' end of the mRNA of mammalian cells is not detectable in Dictyostelium mRNA. Thus neither 2'0-methylated nucleotides nor 6-methyl adenosine can represent sites for processing of a primary nuclear transript to yield mRNA.     

Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification

Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA^Phe, and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxywybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a β-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.     

tadA,an essential tRNA-specific adenosine deaminase from Escherichia coli

We report the characterization of tadA, the first prokaryotic RNA editing enzyme to be identified. Escherichia coli tadA displays sequence similarity to the yeast tRNA deaminase subunit Tad2p. Recombinant tadA protein forms homodimers and is sufficient for site-specific inosine formation at the wobble position (position 34) of tRNA(Arg2), the only tRNA having this modification in prokaryotes. With the exception of yeast tRNA(Arg), no other eukaryotic tRNA substrates were found to be modified by tadA. How ever, an artificial yeast tRNA(Asp), which carries the anticodon loop of yeast tRNA(Arg), is bound and modified by tadA. Moreover, a tRNA(Arg2) minisubstrate containing the anticodon stem and loop is sufficient for specific deamination by tadA. We show that nucleotides at positions 33-36 are sufficient for inosine formation in mutant Arg2 minisubstrates. The anticodon is thus a major determinant for tadA substrate specificity. Finally, we show that tadA is an essential gene in E.coli, underscoring the critical function of inosine at the wobble position in prokaryotes.     

In silico detection of tRNA sequence features characteristic to aminoacyl-tRNA synthetase class membership

Aminoacyl tRNA synthetases (aaRS) are grouped into Class I and II based on primary and tertiary structure and enzyme properties suggesting two independent phylogenetic lineages. Analogously, tRNA molecules can also form two respective classes, based on the class membership of their corresponding aaRS. Although some aaRS-tRNA interactions are not extremely specific and require editing mechanisms to avoid misaminoacylation, most aaRS-tRNA interactions are rather stereospecific. Thus, class-specific aaRS features could be mirrored by class-specific tRNA features. However, previous investigations failed to detect conserved class-specific nucleotides. Here we introduce a discrete mathematical approach that evaluates not only class-specific 'strictly present', but also 'strictly absent' nucleotides. The disjoint subsets of these elements compose a unique partition, named extended consensus partition (ECP). By analyzing the ECP for both Class I and II tDNA sets from 50 (13 archaeal, 30 bacterial and 7 eukaryotic) species, we could demonstrate that class-specific tRNA sequence features do exist, although not in terms of strictly conserved nucleotides as it had previously been anticipated. This finding demonstrates that important information was hidden in tRNA sequences inaccessible for traditional statistical methods. The ECP analysis might contribute to the understanding of tRNA evolution and could enrich the sequence analysis tool repertoire.     

Influence of Soil and Air Temperature on I. Nucleotide Metabolism and Growth of Phaseolus vulgaris L. seedlings

Eight day old greenhouse grown Phaseolus vulgaris L. seedlingswere exposed to three different temperature regimes. The concentrationof the total free nucleotides, nucleoside triphosphates, RNAand protein was much more pronounced in seedlings exposed tothe interactive day/night temperature of 28 ?C (soil) and 15?C (air) of regime I. Seedlings treated to a low soil/air, day/nighttemperature of 15 ?C (regime II) had the lowest content of totalfree nucleotides, nucleoside triphosphates, RNA and protein.The nucleoside triphosphate content and energy charge valueof seedlings treated to a high soil/air, day/night temperatureof 28 ?C (regime III) declined much earlier than in the seedlingsexposed to temperature regimes I and II. The fresh and dry weightof seedlings exposed to temperature regime I was significantlyhigher than those seedlings exposed to the other two temperatureregimes. The slowest growth was observed in seedlings exposedto temperature regime II. The noticeably higher concentrationof ATP, pyridine nucleotides and root nodulation in seedlingsexposed to temperature regime I suggests that higher soil andlow air temperature enhances nitrogen metabolism in P. vulgarisseedlings.     

The nucleotide sequence of rat liver tRNAAsn

The major species of asparagine specific tRNA was isolated from rat liver, degraded to oligonucleotides, and shown to have the nucleotide sequence pG-U-C-U-C-U-G-U-m¹G-m²G-C-G-C- A-A-D-C-G-G-D-X-A-G-C-G-C-m²G-ψ-ψ-C-G-G-C-U-Q-U-U-t⁶A-A-C-C-G- A-A-A-G-m⁷G-D-U-G-G-U-G-G-Z-ψ-C-G-m¹A-G-C-C-C-A-C-C-C-A-G-G-G- A-C-G-C-C-A_OH. Although this tRNA contains several modified nucleotides in their expected positions, it is unique in having X, 3-(3-Amino-3-carboxy-n-propyl)uridine in loop I rather than in loop III; Q, 7-(4,5-cis-dihydroxyl-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine in the wobble position of loop II; and Z, an unknown, and presently uncharacterized nucleoside, at position 23 from the 3′ terminus usually occupied by ribothymidine.     

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.     

Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems

The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.     

Unusual anticodon loop structure found in E.coli lysine tRNA.

Although both tRNA(Lys) and tRNA(Glu) of E. coli possess similar anticodon loop sequences, with the same hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the first position of their anticodons, the anticodon loop structures of these two tRNAs containing the modified nucleoside appear to be quite different as judged from the following observations. (1) The CD band derived from the mnm5s2U residue is negative for tRNA(Glu), but positive for tRNA(Lys). (2) The mnm5s2U monomer itself and the mnm5s2U-containing anticodon loop fragment of tRNA(Lys) show the same negative CD bands as that of tRNA(Glu). (3) The positive CD band of tRNA(Lys) changes to negative when the temperature is raised. (4) The reactivity of the mnm5s2U residue toward H2O2 is much lower for tRNA(Lys) than for tRNA(Glu). These features suggest that tRNA(Lys) has an unusual anticodon loop structure, in which the mnm5s2U residue takes a different conformation from that of tRNA(Glu); whereas the mnm5s2U base of tRNA(Glu) has no direct bonding with other bases and is accessible to a solvent, that of tRNA(Lys) exists as if in some way buried in its anticodon loop. The limited hydrolysis of both tRNAs by various RNases suggests that some differences exist in the higher order structures of tRNA(Lys) and tRNA(Glu). The influence of the unusual anticodon loop structure observed for tRNA(Lys) on its function in the translational process is also discussed.     

The nucleotide sequence of threonine transfer RNA coded by bacteriophage T4.

The nucleotide sequence of a low molecular weight RNA coded by bacteriophage T4 (and previously identified as species alpha) has been determined. The molecule is of particular biological interest for its associated biosynthetic properties. This RNA is 76 nucleotides in length, contains eight modified bases, and can be arranged in a cloverleaf configuration common to tRNAs. The anticodon sequence is UGU, which corresponds to the threonine-specific codons ACA G. The nucleotide sequence was determined primarily by nearest-neighbor analysis of RNA synthesized in vitro using [alpha-32P]nucleoside triphosphates. Using the single-strand specific nuclease S1, two in vivo labeled half-molecules were generated and analysed. This information together with restrictions imposed by nearest-neighbor data, provided a unique linear sequence of nucleotides with the features of secondary structure common to tRNA molecules.     

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.