Enzymatic conversion of guanosine 3'' adjacent to the anticodon of yeast tRNAPhe to N1-methylguanosine and the wye nucleoside: dependence on the anticodon sequence.

N1-Methylguanosine (m1G) or wye nucleoside (Y) are found 3' adjacent to the anticodon (position 37) of eukaryotic tRNAPhe. The biosynthesis of these two modified nucleosides has been investigated. The importance of the type of nucleosides in the anticodon of yeast tRNAPhe on the potentiality of this tRNA to be a substrate for the corresponding maturation enzyme has also been studied. This involved microinjection into Xenopus laevis oocytes and incubation in a yeast extract of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent Y nucleoside were substituted by various tetranucleotides ending with a guanosine. The results obtained by oocyte microinjection indicate: that all the restructured yeast tRNAsPhe are efficient substrates for the tRNA (guanosine-37 N1)methyltransferase. This means that the anticodon sequence is not critical for the tRNA recognition by this enzyme; in contrast, for Y nucleoside biosynthesis, the anticodon sequence GAA is an absolute requirement; the conversion of G-37 into Y-37 nucleoside is a multienzymatic process in which m1G-37 is the first obligatory intermediate; all the corresponding enzymes are cytoplasmic. In a crude yeast extract, restructured yeast tRNAPhe with G-37 is efficiently modified only into m1G-37; the corresponding enzyme is a S-adenosyl-L-methionine-dependent tRNA methyltransferase. The pure Escherichia coli tRNA (guanosine-37 N1) methyltransferase is unable to modify the guanosine-37 of yeast tRNAPhe.     

Photolabile and paramagnetic derivatives of the nucleoside X and of Escherichia coli tRNAPhe.

The synthesis of N3-[3-L-(5-azido-2-nitrobenzamido)-3-carboxypropyl]uridine (4b) and N3-[3-carboxy-3-L-(2,2,5,5-tetramethyl-3-pyrroline-3-carbonylamino)propyl]uridine Npyr-oxyl (4c) starting from the nucleoside X (4a) and the appropriate N-hydroxysuccinimide ester 1 or 2 is described. After acylation of tRNAPhe from E. coli (5a) with 1 or 2, the photolabile tRNAPhe derivative 5b and the paramagnetic tRNAPhe derivative 5c could be isolated. The position of modification in the polynucleotide chain was elucidated by comparison of the ribonuclease II/alkaline phosphatase digestion products of the substituted and unsubstituted tRNAPhe samples, and was identified as being exclusively the amino group of the nucleoside X in position 47 of E. coli tRNAPhe.     

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases from Euglena gracilis. Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126,000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.     

Chloroplastic and cytoplasmic valyl- and leucyl- tRNA synthetases from Euglena gracilis: Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126 000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.     

Synthetic deoxyoligonucleotides as general probes for chloroplast transfer RNA genes

The utility of chemically synthesized deoxyoligonucleotides as hybridization probes for the detection of tRNA genes has been examined. Chloroplast tRNA genes were chosen for this study. Deoxyoligonucleotides complementary to highly conserved regions of chloroplast tRNA genes of both higher plants and Euglena gracilis were chemically synthesized. These synthetic probes have been used to detect tRNA genes by Southern hybridizations to restriction fragments of chloroplast DNAs. This new method of tRNA gene mapping and the oligonucleotides synthesized may be of general application to many chloroplast genomes. This is illustrated by the detection of known and unknown tRNA genes of Euglena gracilis and spinach, and unknown tRNA genes of maize and cucumber chloroplast DNAs. The precise locus and polarity of the Euglena gracilis chloroplast tRNAPhe gene has been determined. We also describe experiments which relate to the effects of the time of hybridization, the stringency of washing, and of base pair mismatches on the hybridization signal.     

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 1. Preparation of yeast tRNAPhe derivatives

The 3'-terminal A-C-C-A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6-ethenoadenosine (epsilon A) or 2-aza-1,N6-ethenoadenosine (aza-epsilon A). T4 RNA ligase was used to join the nucleoside 3',5'-bisphosphates to the 3' end of the tRNA which was shortened by one [tRNAPhe(-A)] or four [tRNAPhe(-ACCA)] nucleotides. It was found that the base-paired 3'-terminal cytidine 72 in tRNAPhe(-ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A-C-C terminus of tRNAPhe(-A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double-stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single-stranded acceptors. The replacement of the 3'-terminal adenosine 76 with epsilon A and aza-epsilon A leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu-specific tRNAs both from yeast and Escherichia coli) are also inactivated by epsilon A incorporation. Replacement of adenosine 73 followed by repair of the C-C-A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of epsilon A and aza-epsilon A at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the epsilon A-labeled tRNAPhe is complexed with phenylalanyl-tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3' terminus is not changed appreciably in these complexes.     

The nucleotide sequence of blue-green algae phenylalanine-tRNA and the evolutionary origin of chloroplasts.

Phenylalanine tRNA from the blue-green alga, Agmenellum quadruplicatum, has been purified to homogeneity. The nucleotide sequence of this tRNA was determined to be: (see tests) Comparisons of the sequence and the modified nucleosides of this tRNA with those of other tRNAPhes thus far sequenced, indicate that this blue green algal tRNAPhe is typically prokaryotic and closely resembles the chloroplast tRNAPhes of higher plants and Euglena. The significance of this observation to the evolutionary origin of chloroplasts is discussed.     

Primary structure of yeast mitochondrial DNA-coded phenylalanine-tRNA.

Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase.     

Crosslinking of N-acetyl-phenylalanyl [s4U]tRNAPhe to protein S10 in the ribosomal P site

In order to identify ribosomal components involved in the peptidyl-tRNA binding site on the ribosome, tRNAPhe molecules were prepared in which cytidine residues had been chemically converted into 4-thiouridine (S4U). This nucleoside is photoactive at 335 nm and able to form covalent bonds with nearby nucleophilic groups. The thiolated AcPhe-tRNAPhe was bound to the ribosomal P site in the presence of poly(U) as verified by puromycin reactivity. Direct irradiation of the AcPhe-[s4U]tRNAPhe poly(U) 70-S ribosome complex induced crosslinking of the tRNA molecule exclusively to 30-S subunits. Analysis of the covalent complex revealed that AcPhe-[s4U]tRNAPhe was specifically crosslinked to protein S10.     

The nucleotide sequence of a cytoplasmic tRNAPhe from Scenedesmus obliquus and comparison with a tRNATyr species.

The nucleotide sequence of a cytoplasmic tRNAPhe from the eukaryotic green alga Scenedesmus obliquus was determined as: pG-G-C-U-U-G-A-U-A-m2G-C-U-C-A-G-C-D-Gm-G-G-A-G-A-G-C-m22G-p si-psi-A-G-A-Cm-U-G - A-A-m1G-A-psi-C-U-A-C-A-G-m7G-N-m5C-C-C-C-A-G-T-psi-C-G-m1A-U-m5C-Cm-U-G -G-G-U- C A-G-G-C-C-A-C-C-A-OH. The structure has some notable features. Unlike other tRNAPhe species from plant sources, it has an unmodified G as the first residue of the anticodon and m1G rather than a Y derivative as the residue following the anticodon. The sequence m5C(60)-Cm(61) is unique to this tRNA. The sequence of S. obliquus tRNAPhe shows close homology with S. obliquus tRNATyr.     

Enzymatic 2''-O-methylation of the wobble nucleoside of eukaryotic tRNAPhe: specificity depends on structural elements outside the anticodon loop.

We have investigated the specificity of the enzyme tRNA (wobble guanosine 2'-O-)methyltransferase which catalyses the maturation of guanosine-34 of eukaryotic tRNAPhe to the 2'-O-methyl derivative Gm-34. This study was done by micro-injection into Xenopus laevis oocytes of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent nucleotide 'Y' were substituted by various tetranucleotides. The results indicate that the enzyme is cytoplasmic; the chemical nature of the bases of the anticodon and its 3' adjacent nucleotide is not critical for the methylation of G-34; the size of the anticodon loop is however important; structural features beyond the anticodon loop are involved in the specific recognition of the tRNA by the enzyme since Escherichia coli tRNAPhe and four chimeric yeast tRNAs carrying the GAA anticodon are not substrates; unexpectedly, the 2'-O-methylation is not restricted to G-34 since C-34, U-34 and A-34 in restructured yeast tRNAPhe also became methylated. It seems probable that the tRNA (wobble guanosine 2'-O-)methyltransferase is not specific for the type of nucleotide-34 in eukaryotic tRNAPhe; however the existence in the oocyte of several methylation enzymes specific for each nucleotide-34 has not yet been ruled out.     

The primary structure of yeast mitochondrial tyrosine tRNA

The mitochondrial tyrosine tRNA from Saccharomyces cerevisiae has been sequenced. It has two interesting structural features: (i) it lacks two semi-invariant purine residues in the D-loop which are involved in tertiary interactions in the yeast cytoplasmic tRNAPhe; (ii) it has a large variable loop and therefore resembles procaryotic tRNAsTyr rather than eucaryotic cytoplasmic ones.     

Compilation of tRNA sequences.

This compilation presents in a small space the tRNA sequences so far published. The numbering of tRNAPhe from yeast is used following the rules proposed by the participants of the Cold Spring Harbor Meeting on tRNA 1978 (1,2;Fig. 1). This numbering allows comparisons with the three dimensional structure of tRNAPhe. The secondary structure of tRNAs is indicated by specific underlining. In the primary structure a nucleoside followed by a nucleoside in brackets or a modification in brackets denotes that both types of nucleosides can occupy this position. Part of a sequence in brackets designates a piece of sequence not unambiguosly analyzed. Rare nucleosides are named according to the IUPACIUB rules (for complicated rare nucleosides and their identification see Table 1); those with lengthy names are given with the prefix x and specified in the footnotes. Footnotes are numbered according to the coordinates of the corresponding nucleoside and are indicated in the sequence by an asterisk. The references are restricted to the citation of the latest publication in those cases where several papers deal with one sequence. For additional information the reader is referred either to the original literature or to other tRNA sequence compilations (3-7). Mutant tRNAs are dealt with in a compilation by J. Celis (8). The compilers would welcome any information by the readers regarding missing material or erroneous presentation. On the basis of this numbering system computer printed compilations of tRNA sequences in a linear form and in cloverleaf form are in preparation.     

Specific phosphorylase from Euglena gracilis splits diadenosine 5',5''-P(1),P(4)-tetraphosphate (Ap(4)A) and diadenosine 5',5''-P(1),P(3)-triphosphate (Ap(3)A)

1. Phosphorolytic cleavage of Ap(4),A was demonstrated in cell-free extracts from two protozoan organisms, Euglena gracilis and Acanthamoeba castellanii. 2. A specific dinucleoside oligophosphate (DNOP) alpha, beta-phosphorylase which degrades substrates with formation of corresponding nucleoside 5'-diphosphate (NDP) as one of the reaction products was purified 625-fold from Euglena gracilis cells. 3. In addition to Ap(4)A, the phosphorylase degrades AP(3)A, Ap(5)A, Gp(4)G and one of phosphonate analogs, ApppCH(2)pA. The K(m) values for Ap(4), A and Ap(3) A are 27 and 25 micron, and relative velocities 100 and 14, respectively. The K(m) for phosphate is 0.5 mM. 4. Some anions (arsenate, chromate, molybdate and vanadate) can substitute for phosphate in the catalyzed reactions and in their presence the DNOPs yield corresponding nucleoside 5'-monophosphate as one of the reactions' product. The enzyme supports also an anion-dependent dephosphorylation of NDPs. 5. Molecular weight of the native Euglena phosphorylase is 30,000. Optimum pH for its activity is at 8.0 Divalent metal cations are essential for the phosphorolysis of DNOPs but are not for the NDP dephosphorylation mentioned.     

Compilation of tRNA sequences.

This compilation presents in a small space the tRNA sequences so far published in order to enable rapid orientation and comparison. The numbering of tRNAPhe from yeast is used as has been done earlier (1) but following the rules proposed by the participants of the Cold Spring Harbor Meeting on tRNA 1978 (2) (Fig. 1). This numbering allows comparisons with the three dimensional structure of tRNAPhe, the only structure known from X-ray analysis. The secondary structure of tRNAs is indicated by specific underlining. In the primary structure a nucleoside followed by a nucleoside in brackets or a modification in brackets denotes that both types of nucleosides can occupy this position. Part of a sequence in brackets designates a piece of sequence not unambiguously analyzed. Rare nucleosides are named according to the IUPAC-IUB rules (for some more complicated rare nucleosides and their identification see Table 1); those with lengthy names are given with the prefix x and specified in the footnotes. Footnotes are numbered according to the coordinates of the corresponding nucleoside and are indicated in the sequence by an asterisk. The references are restricted to the citation of the latest publication in those cases where several papers deal with one sequence. For additional information the reader is referred either to the original literature or to other tRNA sequence compilations (3--7). Mutant tRNAs are dealt with in a separate compilation prepared by J. Celis (see below). The compilers would welcome any information by the readers regarding missing material or erroneous presentation. On the basis of this numbering system computer printed compilations of tRNA sequences in a linear form and in cloverleaf form are in preparation.     

Dihydrouridine-deficient tRNAs in Saccharomyces cerevisiae.

A mutation in Saccharomyces cerevisiae, designated mia, is responsible for the production of isoaccepting tRNA molecules with reduced extents of nucleoside modifications. The mia isoacceptors of tRNAPhe and one of the mutant isoacceptors of tRNATyr were highly purified for nucleoside composition analyses. The data indicate that the mutant isoacceptors are lacking some of the dihydrouridine moieties. This is consistent with our previous hypothesis that the mutant isoacceptors were accumulated due to a defect in a modification process [Lo, R.Y.C. and Bell, J.B. (1981) Current Genetics 3, 73-82). Data from in vitro poly-U translation experiments also support the previous results, suggesting in vivo biological activity of these mutant tRNAs.     

The nucleotide sequence of Euglena cytoplasmic phenylalanine transfer RNA. Evidence for possible classifications of Euglena among the animal rather than the plant kingdom

The nucleotide sequence of cytoplasmic phenylalanine tRNA from Euglena gracilis has been elucidated using procedures described previously for the corresponding chloroplastic tRNA [Cell, 9, 717 (1976)]. The sequence is: pG-C-C-G-A-C-U-U-A-m(2)G-C-U-Cm-A-G-D-D-G-G-G-A-G-A-G-C-m(2)2G-psi-psi-A-G-A-Cm -U-Gm-A-A-Y-A-psi-C-U-A-A-A-G-m(7)G-U-C-*C-C-U-G-G-T-psi-C-G-m(1)A-U-C-C-C-G-G- G-A-G-psi-C-G-G-C-A-C-C-A. Like other tRNA Phes thus far sequenced, this tRNA has a chain length of 76 nucleotides. The sequence of E. gracilis cytoplasmic tRNA Phe is quite different (27 nucleotides out of 76 different) from that of the corresponding chloroplastic tRNA but is surprisingly similar (72 out of 76 nucleotides identical) to that of tRNA Phe from mammalian cytoplasm. This extent of sequence homology even exceeds that found between E. gracilis and wheat germ cytoplasmic tRNA Phe. These findings raise interesting questions on the evolution of tRNAs and the taxonomy of Euglena.     

Competitive inhibition of Phe: tRNA ligase of sheep embryos by aurintricarboxylic acid]

Aurin tricarboxylic acid (A.T.A.), an inhibitor of protein biosynthesis (initiation and elongation steps), acts also as a competitive inhibitor of phenylalanine, in the ATP-PPi exchange and tRNAPhe aminoacylation reactions catalysed by cytoplasmic wheat germ phenylalanine:tRNA ligase.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Chloroplast and Cytoplasmic Ribosomes of Euglena: Selective Binding of Dihydrostreptomycin to Chloroplast Ribosomes

Dihydrostreptomycin binds preferentially to chloroplast ribosomes of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim. The K(diss) for the wild-type chloroplast ribosome-dihydrostreptomycin complex is 2 x 10(-7) M, a value comparable with that found for the Escherichia coli ribosome-dihydrostreptomycin complex. Chloroplast ribosomes isolated from the streptomycin-resistant mutant Sm(1) (r)BNgL and cytoplasmic ribosomes from wild-type have a much lower affinity for the antibiotic. The K(diss) for the chloroplast ribosome-dihydrostreptomycin complex of Sm(1) (r) is 387 x 10(-7) M, and the value for the cytoplasmic ribosome-dihydrostreptomycin complex of the wild type is 1,400 x 10(-7) M. Streptomycin competes with dihydrostreptomycin for the chloroplast ribosome binding site, and preincubation of streptomycin with hydroxylamine prevents the binding of streptomycin to the chloroplast ribosome. These results indicate that the inhibition of chloroplast development and replication in Euglena by streptomycin and dihydrostreptomycin is related to the specific inhibition of protein synthesis on the chloroplast ribosomes of Euglena.