Anticodon Region Research Articles

Large Scale Isolation and Some Properties of AGY-Specific Serine tRNA from Bovine Heart Mitochondria1

A method was developed for large scale isolation of AGY-specific serine tRNA (tRNASerAGY) from bovine heart mitochondria. By this method, 5 A260 units of tRNASerAGY were recovered from 6.3 kg of bovine hearts. The nucleotide sequence was identical to that reported previously. tRNASerAGY showed abnormal melting profiles, as was predicted from its unique primary sequence. Its secondary and/or tertiary structure was analyzed by nuclease digestion method. It was suggested that three extra base pairs could occur in the anticodon stem region, with one adenosine residue protruding. The T loop was quite sensitive to nuclease S1, suggesting that the T loop doesn't interact with other regions. This finding is consistent with the model proposed by Sundaralingam (1980). tRNASerAGY was aminoacylated in vitro with only mitochondrial enzyme but not with the enzymes from E. coli and yeast. The aminoacylation rate of tRNASerAGY with mitochondrial enzyme was much faster than that of cytosolic tRNASerUCN, perhaps reflecting differences due to the presence and absence of the D arm of the tRNAs.

Crystal structure of yeast tRNA Asp: atomic coordinates

The atomic coordinates of yeast aspartic acid transfer RNA, as determined from a crystallographic investigation to 3 A resolution, are presented. In the ribose phosphate backbone sugars are in the C(3')-endo pucker, except for residues A7, A9, D16, G17, G18, D19, C20, U48, A58, and U60 which are in the C(2')-endo pucker. A least-squares superposition of the phosphorus atoms of yeast tRNAAsp and yeast tRNAPhe enlightens both an overall structural similarity and significant conformational differences. The largest deviations occur in the D-loop and the anticodon region.

核酸の機能・構造研究と化学修飾

The use of modified base or alteration of base sequences, and chemical modifications are reviewed for the study of nucleic acid functions and structures. The examples included are the point mutation of promoter region of rDNA gene, replacement of the certain bases with modified ones in the recognition sequence of restriction endonuclease Bgl II and Sau 3AI, ozone-degradation of supercoiled plasmid DNA, replacement of nucleotides in the anticodon region of formylmethionine tRNA, and chemical modification of mouse 5S ribosomal RNA.

A selenium-containing nucleoside at the first position of the anticodon in seleno-tRNAGlu from Clostridium sticklandii.

In previous studies, the single selenonucleoside component of a selenium-containing tRNAGlu isolated from Clostridium sticklandii has been shown to be 5-methyl-aminomethyl-2-selenouridine. Here, we show that this selenonucleoside is most likely located at the "wobble" position of the anticodon of the clostridial seleno-tRNAGlu. Nuclease T1 digestion of this seleno-tRNAGlu generated one major selenium-containing oligonucleotide (25 bases long). The selenium-containing residue within this oligonucleotide was located by sequence analysis of the oligonucleotide before and after removal of selenium by treatment with cyanogen bromide. The sequence of this oligonucleotide, A-A-C-C-G-C-C-C-U-U+-U-C-A+C-G-G-C-G-G-U-A-A-C-A-G, is homologous to that of the Escherichia coli tRNAGlu2 from residues 27 to 50, including the anticodon region and the variable loop, except that the E. coli tRNA has 5-methylaminomethyl-2-thiouridine instead of the selenonucleoside.

UV-Mutagenesis at a cloned target sequence: Converted suppressor mutation is insensitive to mutation frequency decline regardless of the gene orientation

Premutational lesions produced by ultraviolet radiation in the Gln2 tRNA genes of E. coli B/r show differing sensitivities to a mutation avoidance phenomenon known as mutation frequency decline (MFD). A mutation event that changes the wild-type gene to an amber (UAG) suppressor is normally sensitive to MFD. Mutation of this amber suppressor to an ochre (UAA) suppressor is not sensitive to MFD. These two mutation events occur in the same anticodon region of the DNA. The dissimilarity of MFD sensitivity between these two mutations may result because of the respective premutational photoproducts for the two are located in opposite strands of duplex DNA. To examine the effect of strand position of the premutational lesions of MFD, recombinant lambda phage were constructed that contained the amber suppressor as a mutation target in the two possible orientations. Comparison of MFD in bacterial lysogens containing either of the two types of recombinant prophage indicated that reversing the orientation of the target sequence relative to adjacent bacterial DNA had no effect on MFD. Since rotational inversion of the target sequence did not alter the sensitivity to MFD of mutation occurring at the cloned target gene, the antimutation process inherent to FMD can not be attributed to an asymmetrical interaction between the template strands and the DNA-replication complex.

Interaction between dimeric methionyl-tRNA synthetase and methionine accepting tRNAs from E. coli. — Studies by partial ribonuclease digestion

Using different ribonucleases we have studied the digestion pattern of the two methionine accepting tRNAs, the initiator tRNA f Met and the elongator tRNA m Met from E. coli. The positions and intensities of cleavages are compared to those obtained when the tRNAs are complexed to methionyl-tRNA synthetase. Our results, in comparison with other studies, suggest a general pattern of interaction between tRNAs and their cognate synthetases including the amino acid stem and the anticodon region. Furthermore a lack of involvement of the central region and especially the extra arm seems to be a unique feature of the initiator tRNA f Met.

Nucleotide sequence of the split tRNA UAA Leu gene from Vicia faba chloroplasts: evidence for structural homologies of the chloroplast tRNALeu intron with the intron from the autosplicable Tetrahymena ribosomal RNA precursor

The gene encoding the tRNA UAA Leu from broad bean chloroplasts has been located on a 5.1 kbp long BamHI fragment by analysis of the DNA sequence of an XbaI subfragment. This gene is 536 bp long and is split in the anticodon region. The 451 bp long intron shows high sequence homology over about 100 bp from each end with the corresponding regions of the maize chloroplast tRNA UAA Leu intron. These conserved sequences are probably involved in the splicing reaction, for they can be folded into a secondary structure which is very similar to the postulated structure of the intron from the autosplicable ribosomal RNA precursor of Tetrahymena. Very little sequence conservation is found in the 5′-and 3′-flanking regions of the broad bean and maize chloroplast tRNA UAA Leu genes.

Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase.

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl-tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and T psi arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable T psi stem. When alkylation by ethylnitrosourea was studied with the tRNATrp X tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase X tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.

Putative introns in tRNA genes of prokaryotes.

Sequences of two putative tRNA genes, for serine and leucine, from the archaebacterium Sulfolobus solfataricus contain intervening sequences in the anticodon region. Furthermore, the genes lack encoded CCA 3' termini and are flanked by A + T-rich DNA segments. The introns can both form the same secondary structure, which is a double-helical extension of the anticodon stalk. The resulting structure contains two symmetrically placed 3-base bulge loops, in which are located cleavage sites for the introns. In the one case tested, the gene occurs as a single copy in the genome.

Binding of yeast tRNA phe anticodon arm to Escherichia coli 30 S ribosomes

A 15-nucleotide fragment of RNA having the sequence of the anticodon arm of yeast tRNAPhe was constructed using T4 RNA ligase. The stoichiometry and binding constant of this oligomer to poly(U)-programmed 30 S ribosomes was found to be identical to that of deacylated tRNAPhe. The anticodon arm and tRNAPhe also compete for the same binding site on the ribosome. These data indicate that the interaction of tRNAPhe with poly(U)-programmed 30 S ribosomes is primarily a result of contacts in the anticodon arm region and not with other parts of the transfer RNA. Since similar oligomers which cannot form a stable helical stem do not bind ribosomes, a clear requirement for the entire anticodon arm structure is demonstrated.

Degradation of nucleic acids with ozone. III. Mode of ozone-degradation of mouse proline transfer ribonucleic acid (tRNA) and isoleucine tRNA.

The degradation of mouse [32P]proline transfer ribonucleic acid (tRNA) and [32P]isoleucine tRNA with ozone (concentration in inlet gas, 0.1 mg/l) was examined in aqueous solution (reaction time, 0-32 min) and sequence analysis of the products was performed by digestion with ribonuclease A or T1 according to Sanger's method. The guanine moieties in the nucleobases were preferentially attacked by ozone and the cleavage of polynucleotidic linkages did not occur during the ozonization. Proline tRNA was more degradable than isoleucine tRNA, probably as a result of differences in the locations of the guanine residues in their higher-order structures. The consecutive guanine residues in the anticodon and dihydrouracil (D) regions of proline tRNA were most susceptible to degradation with ozone. In the case of isoleucine tRNA, which does not have the conseutive guanine residues in the anticodon region, those in the D-loop, small (S) loop and the common stem were degraded. Therefore ozone seems to start reacting with the guanine moieties located in the most exposed regions of the higher-order structure of tRNA.

Effects of the hisT mutation of Salmonella typhimurium on translation elongation rate.

The hisT mutation in Salmonella typhimurium which results in loss of pseudouridine base modifications in the anticodon regions of many tRNAs was shown to reduce the rate of protein synthesis in vivo by about 20 to 25% as compared with that measured in hisT strains. Reduced protein synthesis rate occurred predominantly at the level of translation rather than transcription. Increased sensitivity of hisT mutants to growth inhibition by antibiotics that inhibit translation elongation, but not by those that inhibit translation initiation, transcription initiation, or transcription elongation, indicates that the hisT mutation leads to a defect in one or more of the steps in the polypeptide chain elongation mechanism. These results can account for effects of the hisT mutation on regulation of certain amino acid biosynthetic operons, including the his, leu, and ilv operons.

Specific interaction of anticodon loop residues with yeast phenylalanyl-tRNA synthetase

Thirteen different yeast tRNAPhe variants with single nucleotide changes in positions 34-37 in the anticodon region were prepared by an enzymatic procedure described previously. Aminoacylation kinetics using purified yeast phenylalanyl-tRNA synthetase revealed that the level of aminoacylation was very different for different sequences inserted. The low level of aminoacylation was the result of a steady state between a slow forward reaction rate and spontaneous deacylation of the product. Aminoacylation kinetics performed at higher synthetase concentrations revealed that substitution at position 34 in tRNAPhe decreased the Km nearly 10-fold but only had a small effect on Vmax. Similar substitutions at positions 35, 36, and 37 had a lesser effect. These data suggest a sequence-specific contact between the anticodon of yeast tRNAPhe and the cognate synthetase.

Enzymatic replacement of the anticodon of yeast phenylalanine transfer ribonucleic acid.

An efficient procedure for the replacement of the anticodon and the adjacent hypermodified nucleotide (residues 34-37) of yeast tRNAPhe with any desired oligoribonucleotide sequence has been developed. The four residues are removed by chemical cleavage at Y-37 and partial ribonuclease A digestion at U-33. An oligonucleotide is inserted in three steps by using T4 RNA ligase and T4 polynucleotide kinase. When different oligonucleotides are inserted, both the size of the loop and the sequence of nucleotides in the anticodon region of this tRNA can be varied. The ability of the different anticodon loop substituted tRNAs to be aminoacylated by yeast phenylalanyl-tRNA synthetase is dependent upon the sequence of the oligonucleotide inserted. This suggests that there is an important interaction between the anticodon region of yeast tRNAPhe and its synthetase.

Purification and properties of a mammalian tRNA pseudouridine synthase.

A tRNA pseudouridine synthase has been extensively purified from steer thymus extracts, using undermodified tRNA from hisT- mutants of Salmonella typhimurium as a substrate. The enzyme synthesizes a group of psi residues in the anticodon region of various hisT- isoacceptors and behaves like a eukaryotic homologue of Salmonella tRNA psi synthase I. The thymus enzyme requires a thiol and a monovalent cation (NH4+ or K+) for optimum activity; no energy sources or cofactors are required. The activity is inhibited by single tRNAs or bulk tRNA from all sources tested, and by ribosomal RNAs, various polyribonucleotides, and DNA. The enzyme modifies the two hisT- tRNAPhe isoacceptors, both tRNATyr acceptors and at least five of the tRNALeu isoacceptors to products that coelute with the respective wild type species on reverse-phase columns. With pure hisT- tRNA2Phe as substrate, the enzyme specifically converts residue U39 to psi. Interestingly, a psi residue is still present at position 32, in the anticodon loop of hisT- tRNA2Phe, indicating the existence of other uncharacterized pseudouridylation enzymes in S. typhimurium. These composite results show that the thymus enzyme can form psi at residues 38, 39, and 40 in the anticodon region of appropriate hisT- isoacceptors. During the enzyme purification, a second activity is partially resolved, which releases 3H from wild type S. typhimurium [pyrimidine-5-3H]tRNA. This activity may be associated with an enzyme that pseudouridylates sites that are uniquely modified in eukaryotic tRNAs, but not in Salmonella tRNAs. Our observations support the view that the psi residues in tRNA are synthesized by a family of enzymes, whose members act on uridine residues in specific regions of the molecule.

Changes of post-transcriptional modification of wye base in tumor-specific tRNAPhe.

Nucleotide sequences of normal mouse liver tRNAPhe and tumor-specific tRNAPhes isolated from Ehrlich ascites tumor and neuroblastoma cells were examined by post-labeling techniques. The results showed that their sequences are identical, except for changes in post-transcriptional modifications that are located in the anticodon region. Normal mouse liver tRNAPhe contained Cm32, Gm34 and YOH37. On the other hand, tumor-specific tRNAPhes were found in one of two possible configurations: 1) Cm32, Gm34 and Y*OH37 (under-modified YOH) or 2) C32, G34 and m1G37. The ratio of the two forms of tRNAPhes differed in different tumor cells; Ehrlich ascites tumor tRNAPhe had mainly Y*OH-containing tRNAPhe whereas neuroblastoma tRNAPhe has predominantly m1G-containing tRNAPhe. It was concluded that tumor-specific tRNAPhes are products of different extents of modification, rather than of new tRNA transcription.

Chemical modification of cytosine residues of tRNAVa1 with hydrogen sulfide (nucleosides and nucleotides. XL).

The conversion of cytosine residue of transfer ribonucleic acid (tRNA) to 4-thiouracil residues was carried out with a hydrogen sulfide-pyridine-water system. In the case of tRNAVal from mouse cells, the cytosine moieties located in unpaired and looped-out regions of the molecule, such as the anticodon and CCA-end regions, were converted. This result shows that the secondary and tertiary structures of tRNA are maintained even in medium containing liquid hydrogen sulfide and pyridine, and hence, this system should be suitable for studies of the higher-order structure of nucleic acids.

A system for measuring the frequencies of tandem and non-tandem double base substitutions induced by ultraviolet irradiation.

A system based on missense suppressor mutations affecting an Escherichia coli glycine tRNA was used for measuring the relative frequencies of a single base substitution, a tandem double base substitution and a non-tandem double base substitution all occurring in the same triplet and in the same mutagenic treatment. RNA sequencing studies reported here have shown that the glyUsuGAA mutation results from a CCC→TTC tandem double substitution in the anticodon region of the gene coding for tRNA GGG Gly . Thus the suGAA allele, like the previously characterized suGAG and suAGA alleles of glyU, affects the tRNA anticodon in the manner anticipated from the coding specificity. The system for measuring relative frequencies of the three suppressor mutations entails the mutagenic treatment of a culture lysogenic for a λ transducing phage carrying glyU+, the wild type tRNA gene. Following mutagenesis the culture is induced for phage production and the resulting lysate assayed for phage carrying any one of the three suppressor alleles. An important feature of the system is that it circumvents the problem of distinguishing the rare double mutations from the more abundant background. We have found that with doses of ultraviolet irradiation sufficient to reduce the viable cell count 98–99%, the tandem double base substitution (suGAA) occurs 0.01 as frequently as the single (suGAG). The tandem double substitution occurs about 4 times more frequently than the nontandem double substitution (suAGA).

Codon binding and translational properties of an isoaccepting lysine tRNA peculiar to virus-transformed Cells.

Isoaccepting lysyl-tRNAs from virus-transformed cells in culture were fractionated in the RPC-5 system into peaks 1, 2, 4, 5a, 5, and 6. tRNALys6 previously was found predominantly associated with transformed cells. The codon response of each peak was determined in an E. coli ribosomal binding assay. tRNALys1, tRNALys2, and tRNALys4 are highly specific for the 5'AAG3' codon. tRNALys5 and tRNALys5a preferentially bind in response to AAA. tRNALys6 binds in response to AAA 3-fold better than in response to AAG. The presence of thiolated nucleosides in the anticodon regions of tRNALys5a, tRNALys5, and tRNALys6 is indicated by I2-inactivation of aminoacylation ability with no effect on the other is isoacceptors. Functional abilities of the isoacceptors were compared in a wheat germ translational system with tobacco mosaic virus RNA as messenger. All of the isoacceptors function about equally well in translation except for tRNALys6, which is only 14 to 24% as effective as the other isoacceptors.

Binding of spermine to tRNATyr stabilizes the conformation of the anticodon loop and creates strong binding sites for divalent cations.

Electron spin resonance (ESR) spectra of yeast tRNATyr. spin-labelled in the isopentenyladenosine residue adjacent to the anticodon, were measured as a function of temperature at various spermine/tRNA ratios. The critical temperature, at which a change in the activation energy for spin-label motion takes place, changes abruptly by almost 10 degrees C upon the addition of the fifth spermine molecule/tRNATyr molecule, indicating a marked stabilization of the anticodon region. Scatchard plots for Mn2+ binding to tRNATyr in the presence of spermine do not follow theoretically predicted curves for electrostatic type of interaction, assuming that four negative charges on tRNA are neutralized by each spermine molecule. It was estimated that two to three new binding sites for divalent cations are created upon the binding of spermine to tRNATyr.