Evaluation of a novel 16S rRNA/tRNA mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)^Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA^Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA^Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA^Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA^Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA^Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.     

Analysis of genomic tRNA revealed presence of novel genomic features in cyanobacterial tRNA

Transfer RNAs (tRNA) are important molecules that involved in protein translation machinery and acts as a bridge between the ribosome and codon of the mRNA. The study of tRNA is evolving considerably in the fields of bacteria, plants, and animals. However, detailed genomic study of the cyanobacterial tRNA is lacking. Therefore, we conducted a study of cyanobacterial tRNA from 61 species. Analysis revealed that; cyanobacteria contain thirty-six to seventy-eight tRNA gens per genome that encodes for 20 tRNA isotypes. The number of iso-acceptors (anti-codons) ranged from thirty-two to forty-three per genome. tRNA^Ile with anti-codon AAU, GAU, and UAU was reported to be absent from the genome of Gleocapsa PCC 73,106 and Xenococcus sp. PCC 7305. Instead, they were contained anti-codon CAU that is common to tRNA^Met and tRNA^Ile as well. The iso-acceptors ACA (tRNA^Cys), ACC (tRNA^Gly), AGA, ACU (tRNA^Ser), AAA (tRNA^Phe), AGG (tRNA^Pro), AAC (tRNA^Val), GCG (tRNA^Arg), AUG (tRNA^His), and AUC (tRNA^Asp) were absent from the genome of cyanobacterial lineages studied so far. A few of the cyanobacterial species encode suppressor tRNAs, whereas none of the species were found to encode a selenocysteine iso-acceptor. Cyanobacterial species encode a few putative novel tRNAs whose functions are yet to be elucidated.     

Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T^? treated with chloramphenicol produces tRNA^phe which is deficient in minor nucleosides. Undermodified tRNA^phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA^phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA^phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA^phe which was similar to the alteration of the purified tRNA^phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA^phe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNA^glu and tRNA^Leu were not changed, but both tRNA^Arg and tRNA^Ile were altered. tRNA₂^Val and tRNA^Met shifted in the elution profile; tRNA₁^Val and tRNA_f^Met were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNA^phe from E. coli 15T^? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA^phe. tRNA^phe from E. coli 15T^?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA^Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.     

Transfer RNA determinants for translational editing by Escherichia coli valyl-tRNA synthetase

Valyl-tRNA synthetase (ValRS) has difficulty differentiating valine from structurally similar non-cognate amino acids, most prominently threonine. To minimize errors in aminoacylation and translation the enzyme catalyzes a proofreading (editing) reaction that is dependent on the presence of cognate tRNA^Val. Editing occurs at a site functionally distinct from the aminoacylation site of ValRS and previous results have shown that the 3′-terminus of tRNA^Val is recognized differently at the two sites. Here, we extend these studies by comparing the contribution of aminoacylation identity determinants to productive recognition of tRNA^Val at the aminoacylation and editing sites, and by probing tRNA^Val for editing determinants that are distinct from those required for aminoacylation. Mutational analysis of Escherichia coli tRNA^Val and identity switch experiments with non-cognate tRNAs reveal a direct relationship between the ability of a tRNA to be aminoacylated and its ability to stimulate the editing activity of ValRS. This suggests that at least a majority of editing by the enzyme entails prior charging of tRNA and that misacylated tRNA is a transient intermediate in the editing reaction.     

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10⁶ daltons (native) DNA.In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA^Val, tRNA₁^Met and tRNA₂^Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm^?3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.     

Temperature dependence of the aminoacylation of tRNA by Bacillus stearothermophilus aminoacyl-tRNA synthetases

Valine specific transfer RNA (tRNA^Val) was isolated from Bacillus stearothermophilus and Escherichia coli by chromatography on benzoylated DEAE–cellulose (BD–cellulose). Likewise isoleucine specific transfer RNA (tRNA^Ile) was isolated from B. stearothermophilus and from Mycoplasma sp. Kid. The thermal denaturation profiles (melting curves) of the two tRNA^Val species in the presence of Mg^{+ +} were nearly identical. However, the T_m for the Kid tRNA^Ile was about 10°C lower than that for the B. stearothermophilus tRNA^Ile. A nuclease and tRNA-free aminoacyl-tRNA synthetase (AA-tRNA synthetase) preparation from B. stearothermophilus was able to function efficiently at temperatures up to 80°C in the aminoacylation of all four tRNA species. Determination of the amino acid-acceptor activity of each tRNA species as a function of temperature of the aminoacylation reaction showed in each case a strong correlation between the loss of acceptor activity and the thermal denaturation profile of the tRNA. Evidence is presented that the loss in acceptor activity is most likely due to a change in structure of the tRNA as opposed to denaturation of the enzyme. These results further support the idea that correct secondary and/or tertiary structure must be maintained for tRNA to be active as a substrate for the AA-tRNA synthetase.     

Interactions of yeast valyl-tRNA synthetase with RNAs and conformational changes of the enzyme.

Different conformations have been identified for the enzyme valyl-tRNA synthetase from yeast inside its complex with one tRNA molecule by neutron scattering. One form is identical to that of the free enzyme in solution; the other form is more contracted, having a radius of gyration which is smaller by 10% and a specific volume which is smaller by 1%. The contracted conformation has been found for the complexes with tRNA^Val and tRNA^Asp in phosphate buffer (pH 6.3) provided the ionic strength is lower than about 150 mm. In higher ionic strength (up to about 500 mm) the enzyme still forms a complex with tRNA^Val but its conformation remains that of the free protein in solution. In the complex with tRNA₃^Leu, the enzyme conformation is that of the free state even at the lowest ionic strength examined (that of the phosphate buffer, 60 mm). The free enzyme is an elongated molecule of radius of gyration 40 Å (a compact protein of the same molecular weight would have a radius of gyration of 30 Å).The positioning within the complex of tRNA^Val, on the one hand, and tRNA₃^Leu, on the other, is very different. The first tRNA is intimately associated with the enzyme, lying predominantly closer to the centre of mass of the complex than the protein. In the complex with tRNA₃^Leu, the tRNA lies further away from the centre of mass of the complex than the protein.Small concentrations of tRNA^Val, tRNA^Asp, tRNA₃^Leu or Escherichia coli 5 S ribosomal RNA cause the enzyme to aggregate into dimers, trimers and higher aggregates provided the ionic strength of the buffer is below 150 mm. In higher ionic strength or for [RNA]: [enzyme] > 1 the aggregates are dissociated to yield the one-to-one RNA-enzyme complex.     

Incorporation of methionine from met-tRNA-Met-F into internal positions of polypeptides by mouse liver polysomes

Two methionine accepting tRNA species corresponding to tRNA_F^Met and tRNA_M^Met from mouse ascites tumor cells were tested for their ability to donate methionine into internal positions of growing polypeptide chains on mouse liver polysomes. Both tRNA species can function in the elongation of polypeptide chains as judged by their ability to incorporate methionine into protein in the absence of chain initiation. The insertion of methionine into internal positions of polypeptide chains from Met-tRNA_F^Met was confirmed by Edman degradation and CNBr cleavage. When both tRNA^Met species were present in saturating concentrations in the cell-free system a strong preference for the incorporation of methionine from Met-tRNA_M^Met became apparent.     

Sequence studies on human placenta tRNAVal : comparison with the mouse myeloma tRNAVal

The major species of valine specific tRNA was isolated from human placenta, degraded to oligonucleotides, and shown to have the nucleotide sequence pG-U-U-U-C-C-G-U-A-G-U-G-U?-A-G-D-G-G-D-D-A-U-C-A-C-m²G-U?-U-C-G-C-C-U-(I or C)-A-C-A-C-G-C-G-A-A-A-G-m⁷G-D-m⁵C-m⁵C-C-C-G-G-U-U?-C-G-m¹A-A-A-C-C-G-G-G-C-G-G-A-A-A-C-A-C-C-A_OH. This human placental tRNA^Val differs from the major species of mouse myeloma tRNA^Val only in that it contains either I or C in the wobble position of the anticodon, and totally lacks 2′-O-methylcytosine and 5-methylcytosine in the anticodon loop.     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

N2-guanine specific transfer RNA methyltransferase II from rat liver

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N²-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m²G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA^Arg, tRNA^Phe, and tRNA^Val yielded significantly more m²G than the bulk tRNA. The K_m for tRNA^Arg in the methylation reaction with enzymes from either tissue was 7.8 × 10⁻⁷ M as compared to the value 1 × 10⁻⁵ M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA^Arg, tRNA^Phe, and tRNA^Val, A-m²G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5′-end contained in a sequence (s⁴)U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary 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 classitics. 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 VIII (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).     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA^Cys, tRNA^Trp, tRNA^Tyr, a major peak of tRNA^Phe, and a large fraction of tRNA^Leu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA^Ser species and with minor tRNA^Leu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.     

Effect of selective chemical modification of 4-thiouridine of phenylalanine transfer ribonucleic acid on enzyme recognition

Transformation of 4-thiouridine residues in Escherichia coli transfer ribonucleic acids is achieved under conditions which leave the major bases and the primary structure unaffected. The modifications of 4-thiouridine involve either alteration with N-ethylmaleimide, cyanogen bromide, or hydrogen peroxide, or a photochemical transformation effected by irradiation at 330 nm of tRNA in an organic solvent. These selective modifications were made on unfractionated species (Phe, Leu, fMet, Tyr, and Val) and purified species (Phe, fMet, and Val) of E. coli tRNA with little or no loss in their capacities to be aminoacylated. Of the tRNA species tested, subsequent treatment of 4-thiouridineless-tRNA with sodium borohydride affects only the capacity of tRNA^Phe to be aminoacylated. These observations are consistent with the proposal that the cognate ligase recognition site on tRNA^Phe is situated in the nonhydrogenbonded dihydrouridine loop area of the molecule.     

Can yeast be used to study mitochondrial diseases? Biolistic tRNA mutants for the analysis of mechanisms and suppressors

Base substitutions equivalent to those causing human pathologies have been introduced in yeast mitochondrial tRNA genes. These mutants can be utilized as flexible tools to investigate the molecular aspects of mitochondrial diseases and identify correcting genes. We show that for all studied tRNA mutations (including an homoplasmic one in tRNA^Val) the severity of phenotypes follows the same trend in four different nuclear backgrounds. Correcting genes include TUF1 and genes encoding aminoacyl-tRNA synthetase. The effect of suppressors was analyzed by Northern blot. Mutated leucyl-tRNA synthetase with highly reduced catalytic activity maintains full suppressing effect, thus suggesting a chaperone-like and/or stabilizing function.     

Rabbit liver tRNAVal1: II. Unusual secondary structure of T ψ C stem and loop due to a U54:A60 base pair

In contrast to all other known tRNAs, mammalian tRNA^Val₁ contains two adenosines A₅₉ and A₆₀, opposite to U₅₄ and ψ₅₅ in the UψCG sequence of the TψC loop, which could form unusual A:U (or A:ψ) pairs in addition to the five “normal” G:C pairs. In order to measure the number of G:C and A:U (A:ψ) pairs in the TψC stem, we prepared the 30 nucleotide long 3′-terminal fragment of this tRNA by “m⁷G-cleavage”. From differentiated melting curves and temperature jump experiments it was concluded that the TψC stem in this fragment is in fact extended by an additional A₆₀:U₅₄ pair. A dimer of this fragment with 14 base pairs was characterized by gel electrophoresis and by the same physical methods. An additional A:U pair in the tRNA^Val₁ fragment does not necessarily mean that this is also true for intact tRNA. However, we showed that U₅₄ is far less available for enzymatic methylation in mammalian tRNA^Val₁ compared to tRNA from T⁻E. coli. This clear difference in U₅₄ reactivity, together with the identification of an extra A₆₀:U₅₄ pair in the UψCG containing fragment suggests the presence of a 6 base pair TψC stem and a 5 nucleotide TψC loop in this tRNA.     

Modified nucleosides and the chromatographic and aminoacylation behavior of tRNAIle from Escherichia coli C6

Transfer RNA from Escherichia coli C6, a Met⁻, Cys⁻, relA⁻ mutant, was previously shown to contain an altered tRNA^Ile which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676–7683). We now report the purification of this altered tRNA^Ile and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA^Ile purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA^Ile (from cysteine-starved cells) was found to have a 5-fold increased V_max in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl ∼ AMP · Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA^Ile bound more efficiently to its synthetase compared to normal tRNA^Ile. Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA^Ile contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA^Ile contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA^Ile without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA^Ile has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA^Ile from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA^Ile for aminoacylation is discussed.