The preparation and properties of a stable mercury derivative of transfer RNAs from Escherichia coli

Further investigations into the properties of the mercury derivative formed by the reaction of 4-thiouridine-containing tRNAs and pentafluorophenylmercury chloride have been carried out. tRNA^fMet (which contains only one 4-thiouridine residue) has been isolated by a one-step column Chromatographic procedure from unfractionated Escherichia coli tRNA and has been shown to react with the mercury compound to give a derivative which has similar properties to those previously reported for the corresponding mercury derivative of tRNA^Tyr which contains two adjacent 4-thiouridine residues. The mercury derivative of tRNA^Tyr appears to be a competitive inhibitor of tRNA^Tyr in the aminoacylation reaction (tRNA^TyrK_m = 0.42 μM, mercury derivative of tRNA^TyrK_i = 0.11 μM). The mercury derivative of Tyr-tRNA^Tyr can be made, but only by the reaction of the mercury compound with the aminoacylated tRNA.     

Conformation effects of base modification on the anticodon stem-loop of Bacillus subtilis tRNA(Tyr)

tRNA molecules contain 93 chemically unique nucleotide base modifications that expand the chemical and biophysical diversity of RNA and contribute to the overall fitness of the cell. Nucleotide modifications of tRNA confer fidelity and efficiency to translation and are important in tRNA-dependent RNA-mediated regulatory processes. The three-dimensional structure of the anticodon is crucial to tRNA-mRNA specificity, and the diverse modifications of nucleotide bases in the anticodon region modulate this specificity. We have determined the solution structures and thermodynamic properties of Bacillus subtilis tRNA^Tyr anticodon arms containing the natural base modifications N⁶-dimethylallyl adenine (i⁶A₃₇) and pseudouridine (ψ₃₉). UV melting and differential scanning calorimetry indicate that the modifications stabilize the stem and may enhance base stacking in the loop. The i⁶A₃₇ modification disrupts the hydrogen bond network of the unmodified anticodon loop including a C₃₂-A₃₈⁺ base pair and an A₃₇-U₃₃ base-base interaction. Although the i⁶A₃₇ modification increases the dynamic nature of the loop nucleotides, metal ion coordination reestablishes conformational homogeneity. Interestingly, the i⁶A₃₇ modification and Mg²⁺ are sufficient to promote the U-turn fold of the anticodon loop of Escherichia coli tRNA^Phe, but these elements do not result in this signature feature of the anticodon loop in tRNA^Tyr.     

The tRNATyr multigene family of Nicotiana rustica: genome organization,sequence analyses and expression in vitro

Tobacco tRNA^Tyr genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNA^Tyr-specific probe to Southern blots of Eco RI-digested DNA. A Nicotiana genomic library was prepared by Eco RI digestion of nuclear DNA, ligation of the fragments into the vector gtWES·B and in vitro packaging. The phage library was screened with a 5-labelled synthetic oligonucleotide complementary to nucleotides 18 to 37 of cytoplasmic tobacco tRNA^Tyr. Eleven hybridizing Eco RI fragments ranging in size from 1.7 to 7.5 kb were isolated from recombinant lambda phage and subcloned into pUC19 plasmid. Four of the sequenced tRNA^Tyr genes code for the known tobacco tRNA₁ ^Tyr (GA) and seven code for tRNA₂ ^Tyr (GA). The two tRNA species differ in one nucleotide pair at the basis of the TC stem. Only one tRNA^Tyr gene (pNtY5) contains a point mutation (T54A54). Comparison of the intervening sequences reveals that they differ considerably in length and sequence. Maturation of intron-containing pre-tRNAs was studied in HeLa and wheat germ extracts. All pre-tRNAs^Tyr-with one exception-are processed and spliced in both extracts. The tRNA^Tyr gene encoded by pNtY5 is transcribed efficiently in HeLa extract but processing of the pre-tRNA is impaired.     

Structural Changes of Yeast tRNATyr Caused by the Binding of Divalent Ions in the Presence of Spermine

Abstract

The existence of specific sites in tRNA for the binding of divalent cations has been seriously questioned by electrostatic considerations [Leroy & Guéron (1979) Biopolymers, 16, 2429–2446], However, our earlier studies of the binding of Mg²⁺ and Mn²⁺ to yeast tRNA^Tyr have indicated that spermine creates new binding sites for divalent cations [Weygand-Durasevi? et al. (1977) Biochim. Biophys. Acta, 479, 332–344; Nöthig-Laslo et al. (1981) Eur. J. Biochem. 117, 263–267]. We have now used yeast tRNA^Tyr, spin labeled at the hypermodified purine (i⁶A-37) in the anticodon loop, to study the effect of spermine on the binding of manganese ions. The presence of eight spermine molecules per tRNA^Tyr at high ionic strength (0.2 M NaCl, 0.05 M triethanolamine-HCl) and at low temperature (7°C) enhances the binding of manganese to tRNA^Tyr. This effect could not be explained by electrostatic binding. The initial binding of manganese to tRNA^Tyr affects the motional properties of the spin label indicating a change of the conformation of the anticodon loop. From the absence of the paramagnetic effect of manganese on the ESR spectra of the spin label one can conclude that the first binding site for manganese is at a distance from i⁶A-37, influencing the spin label motion through a long-range effect. The enhancement of the binding of manganese to tRNA^Tyr by spermine is lost upon destruction of its specific macromolecular structure and it does not occur in single stranded or in double-stranded polynucleotides. The observed effect can be explained by the binding of Mn²⁺ to new sites, created by the binding of spermine, which are specific for the macromolecular structure of tRNA.     

Isoaccepting mitochondrial glutamyl-tRNA species transcribed from different regions of the mitochondrial genome of Saccharomyces cerevisiae

Mitochondrial glutamyl-tRNA isolated from mitochondria of Saccharomyces cerevisiae was separated into two distinct species by re versed-phase chromatography. The migration of the two mitochondrial glutamyl-tRNAs (tRNA_I^Glu and tRNA_II^Glu) differed from that of two glutamyl-tRNA species found in the cytoplasm of a mitochondrial DNA-less petite strain. Both mitochondrial tRNAs hybridized with mitochondrial DNA. Three lines of evidence demonstrate that mitochondrial tRNA_I^Glu and tRNA_II^Glu are transcribed from different mitochondrial cistrons. First the level of hybridization of a mixture of the two tRNAs to mitochondrial DNA was equal to the sum of the saturation hybridization levels of each glutamyl-tRNA alone. Second, the two mitochondrial glutamyl-tRNAs did not compete with each other in hybridization competition experiments. Finally the tRNAs showed individual hybridization patterns with different petite mitochondrial DNAs.Hybridization of the tRNAs to mitochondrial DNA of genetically defined petite strains localized each tRNA with respect to antibiotic resistance markers. The two glutamyl-tRNA cistrons were spatially separated on the genetic map.     

The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts

Translation of tobacco mosaic virus (TMV) RNA in tobacco protoplasts yields the 17.5-K coat protein, a 126-K protein and a 183-K protein which is generated by an efficient readthrough over the UAG termination codon at the end of the 126-K cistron. In wheat germ extracts, however, only the 5'-proximal 126-K cistron is translated whereas the 183-K readthrough protein is not synthesized. Purification and sequence analysis of the endogenous tyrosine tRNAs revealed that the uninfected tobacco plant contains two tRNAs^Tyr, both with GΨA anticodons which stimulate the UAG readthrough in vitro and presumably in vivo. In contrast, ˜85% of the tRNA^Tyr from wheat germ contains a QΨA anticodon and ˜15% has a GΨA anticodon. Otherwise the sequences of tRNAs^Tyr from wheat germ and tobacco are identical. UAG readthrough and hence synthesis of the 183-K protein is only stimulated by tRNA^Tyr_GΨA and not at all by tRNA^Tyr_QΨA. The tRNAs^Tyr from wheat leaves were also sequenced. This revealed that adult wheat contains tRNA^Tyr_GΨA only. This is very much in contrast to the situation in animals, where Q-containing tRNAs are characteristic for adult tissues whereas Q deficiency is typical for the neoplastic and embryonic state.     

Discrimination between transfer-RNAs by tyrosyl-tRNA synthetase

We have constructed a model of the complex between tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and tRNA^Tyr by successive cycles of predictions, mutagenesis of TyrRS and molecular modeling. We confront this model with data obtained independently, compare it to the crystal structures of other complexes and review recent data on the discrimination between tRNAs by TyrRS. Comparison of the crystal structures of TyrRs and GlnRS, both of which are class I synthetases, and comparison of the identity elements of tRNA^Tyr and tRNA^Gln indicate that the two synthetases bind their cognate tRNAs differently. The mutagenesis data on tRNA^Tyr confirm the model of the TyrRS:tRNA^Tyr complex on the following points. TyrRS approaches tRNA^Tyr on the side of the variable loop. The bases of the first three pairs of the acceptor stem are not recognized. The presence of the NH₂ group in position C6 and the absence of a bulky group in position C2 are important for the recognition of the discriminator base A73 by TyrRS, which is fully realized only in the transition state for the acyl transfer. The anticodon is the major identity element of tRNA^Tyr. We have set up an in vivo approach to study the effects of synthetase mutations on the discrimination between tRNAs. Using this approach, we have shown that residue Glul52 of TyrRS acts as a purely negative discriminant towards non-cognate tRNAs, by electrostatic and steric repulsions. The overproductions of the wild type TyrRSs from E coli and B stearothermophilus are toxic to E coli, due to the mischarging or the non-productive binding of tRNAs. The construction of a family of hybrids between the TyrRSs from E coli and B stearothermophilus has shown that their sequences and structures have remained locally compatible through evolution, for holding and function, in particular for the specific recognition and charging of tRNA^Tyr.     

Relative stabilities of RNA/DNA hybrids: effect of RNA chain length in competitive hybrization

Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA^Tyr-like sequences transcribed in vitro on φ80psu₃⁺ bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA^Tyr hybridization revealed that the in vitro-synthesized RNA contained significant amounts of tRNA^Tyr; these sequences could not, however, be detected by the direct competition method in which labelled in vitro-synthesized RNA competes with E. coli tRNA^Tyr for hybridization to φ80psu₃⁺ DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro-transcribed tRNA^Tyr-like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA^Tyr from its hybrids with φ80psu₃⁺ DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA^Tyr.     

Influence of the A15 mutation on the conformational energy balance in Escherichia coli tRNA Tyr.

The A15 mutation in Escherichia coli tRNA_su3^Tyr produces a transfer RNA whose tertiary structure has either a higher or lower t_m than the wild type, depending on Mg²⁺ concentration. The enthalpy that stabilizes the tertiary structure is greatly reduced by the A15 mutation, but there are large compensating entropy changes. At 37 °C the mutation decreases the magnitude of the free energy stabilizing the tertiary structure for all Mg²⁺ concentrations. The nucleotide modifications s⁴U, iA and G¹ do not contribute detectably to tertiary structure stability. The results can be interpreted in terms of a tertiary pairing between A15 and C57 in tRNA_{su3 A15}^Tyr, of a form suggested by the unusual bonding between G15 and C48 found in crystallographic studies of yeast tRNA^phe. The observed disturbance in the conformational energy balance should contribute to the defective function of tRNA_{su3 A15}^Tyr.     

Physical mapping of the transfer RNA genes on lambda-h80dglytsu+36

The three Escherichia coli transfer RNA genes of the DNA of the transducing phage λ80cI857S^?t68dglyTsu⁺₃₆tyrTthrT (abbreviated λh80T), which specify the structures of tRNA^Gly₂(su⁺₃₆), tRNA^Tyr₂ and tRNA^Thr₃, have been mapped by hybridizing ferritin-labeled E. coli tRNA to heteroduplexes of λh80T DNA with the DNA of the parental phage (λh80cI857S^?t68) and examining the product in the electron microscope. The DNA of λh80T contains a piece of bacterial DNA of length 0·43 λ unit³ that replaces a piece of phage DNA of length 0·46 λ unit, proceeding left from B · P′ (the junction of bacterial DNA and phage DNA) (i.e. att⁸⁰). A cluster of three ferritin binding sites, and thus of tRNA genes, is seen at a position of 0·24 λ unit (1·1 × 10⁴ nucleotides) to the left of B· P′. The three tRNA genes of the cluster are separated by the unequal spacings of 260 (±30) and 140 (± 30) nucleotides, proceeding left from B·P′. The specific map positions have been identified by hybridization competition between ferritin-labeled whole E. coli tRNA with unlabeled purified tRNA^Tyr₂ and with unlabeled partially purified tRNA^Gly₂. The central gene of the cluster is tRNA^Tyr₂. The tRNA^Gly₂gene is probably the one furthest from B·P′. Thus, the gene order and spacings, proceeding left from B·P′, are: tRNA^Thr₃, 260 nucleotides, tRNA^Try₂, 140 nucleotides, tRNA^Gly₂.     

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

N⁶-Threonylcarbamoyl-adenosine (t⁶A) is a universal modification occurring at position 37 in nearly all tRNAs that decode A-starting codons, including the eukaryotic initiator tRNA (tRNA_i^Met). Yeast lacking central components of the t⁶A synthesis machinery, such as Tcs3p (Kae1p) or Tcs5p (Bud32p), show slow-growth phenotypes. In the present work, we show that loss of the Drosophila tcs3 homolog also leads to a severe reduction in size and demonstrate, for the first time in a non-microbe, that Tcs3 is required for t⁶A synthesis. In Drosophila and in mammals, tRNA_i^Met is a limiting factor for cell and animal growth. We report that the t⁶A-modified form of tRNA_i^Met is the actual limiting factor. We show that changing the proportion of t⁶A-modified tRNA_i^Met, by expression of an un-modifiable tRNA_i^Met or changing the levels of Tcs3, regulate target of rapamycin (TOR) kinase activity and influences cell and animal growth in vivo. These findings reveal an unprecedented relationship between the translation machinery and TOR, where translation efficiency, limited by the availability of t⁶A-modified tRNA, determines growth potential in eukaryotic cells.     

Cloning of the yeast tyrosine transfer RNA genes in bacteriophage lambda.

Lambda bacteriophage containing yeast tyrosine transfer RNA genes were prepared by molecular recombination. These phage were identified by hybridization of ¹²⁵I-labeled yeast tRNA^Tyr to plaques from lambda-yeast recombinant phage pools. The cloned yeast EcoRI fragments that hybridize to ¹²⁵I-labeled tRNA^Tyr were compared in size with the fragments in total yeast DNA that hybridize to the same probe. These comparisons indicate that seven of the eight different tRNA^Tyr genes have been isolated. Unambiguous evidence that these seven fragments contain tRNA^Tyr coding regions was obtained by showing that they hybridize to aminoacylated [^3H]Tyr-tRNA^Tyr. Only one of the fragments hybridizes to ³²P-labeled total yeast tRNA in the presence of competing unlabeled tRNA^Tyr; the tRNA^Tyr genes, therefore, are not predominantly organized into heteroclusters of tRNA genes.     

The genes coding for tRNATyr of Drosophila melanogaster: Localization and determination of the gene numbers

Transfer RNA^Tyr (anticodon GA) was isolated from Drosophila melanogaster by means of Sepharose 4B, RPC-5, and polyacrylamide gel electrophoresis. The tRNA was iodinated in vitro with Na¹²⁵I and hybridized in situ to salivary gland chromosomes from Drosophila. The genes of tRNA^Tyr were localized in eight regions of the genome by autoradiography. Restriction enzyme analysis of genomic DNA indicated that the haploid Drosophila genome codes for about 23 tRNA^Tyr genes. The regions 22F and 85A each contain four to five tRNA^Tyr genes, whereas the regions 28C, 41AB, 42A, 42E, and 56D each contain two to three tRNA^Tyr genes.     

Mitochondrial tyrosyl transfer ribonucleic Acid in soybean seedlings

Soybean seedlings were examined for the presence of mitochondrial tRNA. Tyrosyl transfer tRNAs from whole cells, from a well characterized mitochondrial preparation, and from a snake venom phosphodiesterase-treated mitochondrial preparation, were compared by reverse phase chromatography. It was concluded that none of the three previously reported tRNA^Tyr species were mitochondrial. Rather, a fourth tRNA^Tyr species, eluting somewhat later, was of mitochondrial origin. Mitochondrial tRNA^Tyr was chromatographically similar to Escherichia coli tRNA^Tyr.     

Nuclear tRNATyr genes are highly amplified at a single chromosomal site in the genome of Arabidopsis thaliana

Summary We have examined the organization of tRNA^Tyr genes in three ecotypes of Arabidopsis thaliana, a plant with an extremely small genome of 7 × 107 bp. Three tRNA^Tyr gene-containing EcoRI fragments of 1.5 kb and four fragments of 0.6, 1.7, 2.5 and 3.7 kb were cloned from A. thaliana cv. Columbia (Col-O) DNA and sequenced. All EcoRl fragments except those of 0.6 and 2.5 kb comprise an identical arrangement of two tRNA^Tyr genes flanked by a tRNA^Ser gene. The three tRNA genes have the same polarity and are separated by 250 and 370 bp, respectively. The tRNA^Tyr genes encode the known cytoplasmic tRNA_GA ^Tyr. Both genes contain a 12 by long intervening sequence. Densitometric evaluation of the genomic blot reveals the presence of at least 20 copies, including a few multimers, of the 1.5 kb fragment in Col-O DNA, indicating a multiple amplification of this unit. Southern blots of EcoRl-digested DNA from the other two ecotypes, cv. Landsberg (La-O) and cv. Niederzenz (Nd-O) also show 1.5 kb units as the major hybridizing bands. Several lines of evidence support the idea of a strict tandem arrangement of this 1.5 kb unit: (i) Sequence analysis of the EcoRI inserts of 2.5 and 0.6 kb reveals the loss of an EcoRI site between 1.5 kb units and the introduction of a new EcoRI site in a 1.5 kb dimer. (ii) Complete digestion of Col-O DNA with restriction enzymes which cleave only once within the 1.5 kb unit also produces predominantly 1.5 kb fragments. (iii) Partial digestion with EcoRI shows that the 1.5 kb fragments indeed arise from the regular spacing of the restriction sites. The high degree of sequence homology among the 1.5 kb units, ranging from 92% to 99%, suggests that the tRNA^Ser/tRNA^Tyr cluster evolved 1–5 million years ago, after the Brassicaceae diverged from the other flowering plants about 5–10 million years ago.     

Changes in Rhodopseudomonas sphaeroides isoaccepting phenylalanyl-tRNA species during transitions from chemoheterotrophic to photoheterotrophic growth

A new iso-accepting tRNA^phe from extracts of chemoheterotrophic and photoheterotrophic cells of Rhodopseudomonas sphaeroides has been identified by both BDEAE cellulose and RPC-5 chromatography. Rechromatography of each of the tRNA^phe species in either the acylated or deacylated state shows that they migrate as single homogeneous peaks.In steady-state chemoheterotrophic cultures of R. sphaeroides tRNA _I–II ^phe account for 25–30% of the total phenylalanine accepting activity while in steadystate photoheterotrophic cultures tRNA _I–II ^phe account for no more than 10% of the total phenylalanine accepting activity.During the transition from chemoheterotrophic to photoheterotrophic growth conditions the levels of tRNA _I–II ^phe fall in an exponential manner during the first half of the intracytoplasmic membrane induction period. tRNA _I ^phe then remains at a level 10% that of its steady-state chemoheterotrophic level as long as photoheterotrophic growth conditions remain. tRNA _II ^phe , after dropping to 10% of its former chemoheterotrophic level then returns to a level 50% that of its chemoheterotrophic level as long as photoheterotrophic growth conditions remain.Abbreviations BDEAE benzoylated diethyl amino ethyl - RPC reversed phase chromatography - TCA tricholroacetic acid - ICM intracytoplasmic membrane Submitted by WDS in partial fullfilment of requirements for the M.S. degree     

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA^Tyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA^Tyr(GΨA). Structural information for TyrRS–tRNA^Tyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNA^Tyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA^Tyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA^Tyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA^Tyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.     

The anticodon-anticodon complex

Gel electrophoresis has been used to measure the binding between two tRNAs with complementary anticodons, tRNA^Val (Escherichia coli) (anticodon X,A,C) and tRNA^Tyr (E. coli) (anticodon Q,U,A). The association constant K at 0 °C was found to be 4 × 10⁵ m^?1 which is about three orders of magnitude greater than the association constant for tRNA^Tyr (E. coli) binding its trinucleotide codon UAC. The temperature dependence of K suggests that this results from the rigidity of the anticodon loop. tRNA^Tyr (E. coli) binds an order of magnitude more weakly to tRNA^Val (yeast) than to tRNA^Val (E. coli), presumably because it contains the wobble base pair A · I. The relationship between the anticodon-anticodon complex and codon recognition is discussed.