Position Of Anticodon Research Articles

The Difference in the Type of Codon-Anticodon Base Pairing at the Ribosomal P-Site Is One of the Determinants of the Translational Rate1

By utilizing an enzymatically reconstructed tRNA variant containing an altered anticodon sequence, we have examined the different biochemical behavior of translation between the Watson-Crick type and the wobble type base pair interactions at the first anticodon position. We have found that the Watson-Crick type base pair has an advantage in translation in contrast to the wobble type base pair by comparing the efficiency of transpeptidation of native tRNA(Phe) (anticodon; GmAA) with its variant tRNA (anticodon; AAA) in the poly(U)-programmed ribosome system. Thomas et al. [Proc. Natl. Acad. Sci. U.S. (1988) 85, 4242-4246] showed that the wobble codon at the ribosomal A-site accepted its cognate tRNA less efficiently than the Watson-Crick base pairing codon. We report here that the wobble interaction at the ribosomal P-site also affected the rate of translation. This variable translational rate may be a mechanism of gene regulation through preferential codon usage.

Glycine tRNA mutants with normal anticodon loop size cause -1 frameshifting.

Mutations in the acceptor stem, the 5-methyluridine-pseudouridine-cytidine (TFC) arm, and the anticodon of Salmonella tRNA2Gly can cause -1 frameshifting. The potential for standard base pairing between acceptor stem positions 1 and 72 is disrupted in the mutant sufS627. This disruption may interfere with the interaction of the tRNA with elongation factor-Tu.GTP or an as-yet-unspecified domain of the ribosome. The potential for standard base pairing in part of the TFC stem is disrupted in mutant sufS625. The nearly universal C-61 base of the TFC stem is altered in mutant sufS617, and the TFC loop is extended in mutant sufS605. These changes are expected to interfere with the stability of the TFC loop and its interaction with the D arm. The mutation in mutant sufS605, and possibly other mutants, alters nucleoside modification in the D arm. Three mutants, sufS601, sufS607, and sufS609, have a cytidine substituted for the modified uridine at position 34, the first anticodon position. None of the alterations grossly disrupts in-frame triplet decoding by the mutant tRNAs. The results show that -1 frameshifting in vivo can be caused by tRNAs with normal anticodon loop size and suggest that alternative conformational states of the mutant tRNAs may allow them to read a codon in frame or to shift reading frame.

A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium (lysidine), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

Occurrence of unmodified adenine and uracil at the first position of anticodon in threonine tRNAs in Mycoplasma capricolum.

Codon usage pattern in the threonine four-codon (ACN) box in Mycoplasma capricolum is strongly biased towards adenine and uracil for the third base of codons. Codons ending in uracil or adenine, especially ACU, predominate over ACC and ACG. This bacterium contains two isoacceptor threonine tRNAs having anticodon sequences AGU and UGU, both with unmodified first nucleotides. It would thus appear that ACN codons are translated in an unusual way; tRNA(Thr)(AGU) would translate the most abundantly used codon ACU exclusively, because adenine at the first anticodon position can, according to the wobble rule, pair only with uracil of the third codon position. The tRNA(Thr)(UGU) would mainly be responsible for translation of three other codons, ACA, ACG, and ACC. Anticodon UGU would also be used for reading codon ACU as a redundancy of tRNA(Thr)-(AGU), as deduced from the mitochondrial code where unmodified uracil at the first anticodon position can pair with adenine, cytosine, guanine, and uracil by four-way wobble. The tRNA(Thr)(AGU) has much higher sequence homology to tRNA(Thr)(UGU) from M. capricolum (88%), Bacillus subtilis (77%) and Escherichia coli (86%) than to tRNA(Thr)(GGU) from B. subtilis (66%) and E. coli (63%), suggesting that tRNA(Thr)-(AGU) has been derived from tRNA(Thr)(UGU), but not from tRNA(Thr)(GGU).

Structure and expression of the genes, mcrBDCGA , which encode the subunits of component C of methyl coenzyme M reductase in Methanococcus vannielii

The genes that encode the alpha, beta, and gamma subunits of component C of methyl coenzyme M reductase (mcrA, mcrB, and mcrG) in Methanococcus vannielii have been cloned and sequenced, and their expression in Escherichia coli has been demonstrated. These genes are organized into a five-gene cluster, mcrBDCGA, which contains two genes, designated mcrC and mcrD, with unknown functions. The mcr genes are separated by very short intergenic regions that contain multiple translation stop codons and strong ribosomebinding sequences. Although the genome of M. vannielii is 69 mol% A+T, there is a very strong preference in the mcrA, mcrB, and mcrG genes for the codon with a C in the wobble position in the codon pairs AA(U) (C) (asparagine), GA(U) (C) (aspartic acid), CA(U) (C) (histidine), AU(U) (C) (isoleucine), UU(U) (C) (phenylalanine), and UA(U) (C) (tyrosine). The mcrC and mcrD genes do not show this codon preference and frequently have U or A in the wobble position. As the codon pairs listed above are likely to be translated by the same tRNA with a G in the first anticodon position, the presence of C in the wobble position might ensure maximum efficiency of translation of transcripts of these very highly expressed genes.

Antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of Schizosaccharomyces pombe.

Antisuppressor mutations reduce the efficiency of nonsense suppressors. A mutation in the gene sin4 of Schizosaccharomyces pombe leads to loss of 5-(methoxycarbonylmethyl) thiouridine (mcm5s2U) from the first anticodon position of tRNAs. This resembles the phenotype of sin3 (Heyer, W. D., Thuriaux, P., Kohli, J., Ebert, P., Kersten, H., Gehrke, C., Kuo, K. C., and Agris, P. F. (1984) J. Biol. Chem. 259, 2856-2862), but the mutations reside in different genes. In vivo 35S-labeled tRNA from the parental suppressor strain sup3, the antisuppressor strains sin3 and sin4, and the double mutant sin3 sin4 has been digested to nucleosides and analyzed with high performance liquid chromatography methods. The major sulfur-carrying nucleoside in wild-type S. pombe tRNA is mcm5s2U. It is reduced in the mutant strains. Two other thiolated nucleosides are also present: 2-thiouridine and a nucleoside of unknown structure. Neither was affected by the antisuppressor mutations. Thiocytidine has not been found. Independent from their effect on suppressors, the two mutations sin3 and sin4 reduce the growth rate of cells, and sin3 also increases cell length. In vivo decoding of the serine codon UCG by the UCA reading serine tRNA is not promoted by the two antisuppressor mutations.

Specific substitution into the anticodon loop of yeast tyrosine transfer RNA

The aminoacylation kinetics of 19 different variants of yeast tRNATyr with nucleotide substitutions in positions 33-35 were determined. Substitution of the conserved uridine-33 does not alter the rate of aminoacylation. However, substitution of the anticodon position 34 or position 35 reduces Km from 2- to 10-fold and Vmax as much as 2-fold, depending on the nucleotide inserted. The ochre and amber suppressor tRNAsTyr both showed about a 7-fold reduction in Vmax/Km. Data from tRNATyr with different modified nucleotides at position 35 suggest that specific hydrogen bonds form between the synthetase and both the N1 and N3 hydrogens of psi-35. The effect of simultaneous substitutions at positions 34 and 35 can be predicted reasonably well by combining the effects of single substitutions. These data suggest that yeast tyrosyl-tRNA synthetase interacts with positions 34 and 35 of the anticodon of tRNATyr and opens the possibility that nonsense suppressor efficiency may be mediated by the level of aminoacylation.

Construction of a systematic set of tRNA mutants by ligation of synthetic oligonucleotides into defined single-stranded gaps.

A series of mutant tRNA genes has been constructed by site-directed mutagenesis in pOP203, a colE1 derivative carrying a transcription unit under control of the lacUV5 promoter. These mutant genes include all possible amber suppressing variants of tRNATrp with single nucleotide substitutions at anticodon loop positions 32, 37, and 38 (numbered from the 5' end), and all possible paired base substitutions in the three base pairs nearest the anticodon loop. G at position 38 was not recovered as a single mutation, but rather in conjunction with an undirected mutation to T at position 32. The singly mutated G38 tRNA may not be active, though all the other tRNA derivatives are functional in the translation of amber codons. To construct the mutants, we ligated a synthetic deoxyoligonucleotide into a precisely formed single-stranded gap covering the anticodon arm region DNA, in an otherwise double-stranded fragment containing the tRNATrp gene. The resulting heteroduplex was then ligated into the plasmid and introduced into Escherichia coli. This method of mutagenesis is simple, reproducible, and highly tolerant of varying degrees of heteroduplex in the gap, variations in temperature of ligation, and changes in the oligonucleotide concentration. Mutagenesis does not require a 5'-phosphorylated oligonucleotide. These qualities suit the gap method for intensive study of a region by site-directed mutagenesis.

A multivariate study of the relationship between the genetic code and the physical-chemical properties of amino acids

The 20 naturally occurring amino acids are characterized by 20 variables: pKNH2, pKCOOH, pI, molecular weight, substituent van der Waals volume, seven 1H and 13C nuclear magnetic resonance shift variables, and eight hydrophobicity-hydrophilicity scales. The 20-dimensional data set is reduced to a few new dimensions by principal components analysis. The three first principal components reveal relationships between the properties of the amino acids and the genetic code. Thus the amino acids coded for by adenosine (A), uracil (U), or cytosine (C) in their second codon position (corresponding to U, A, or G in the second anticodon position) are grouped in these components. No grouping was detected for the amino acids coded for by guanine (G) in the second codon position (corresponding to C in the second anticodon position). The results show that a relationship exists between the physical-chemical properties of the amino acids and which of the A (U), U (A), or C (G) nucleotide is used in the second codon (anticodon) position. The amino acids coded for by G (C) in the second codon (anticodon) position do not participate in this relationship.

Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons. The effect of modified bases adjacent to the anticodon triplet.

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.

Anticodon-anticodon interactions in solution: Studies of the self-association of yeast or Escherichia coli tRNA Asp and of their interactions with Escherichia coli tRNA Val

The temperature-jump method was used to measure the thermodynamic and kinetic parameters of the yeast tRNA Asp (anticodon GUC) duplex, which involves a U/U mismatch in the middle position of the quasi self-complementary anticodon, and of the yeast tRNA Asp (GUC)- Escherichia coli tRNA Val (GAC) complex, in which the tRNAs have complementary anticodons. The existence of the tRNA Asp duplex involving GUC-GUC interactions as evidenced in the crystal structure has now been demonstrated in solution. However, the value of its association constant ( K ass = 10 4 m −1 at 0 °C) is characteristic of a rather weak complex, when compared with that between tRNA Asp and tRNA Val ( K ass = 4 × 10 6 m −1 at 0 °C), the effect being essentially linked to differences in the rate constant for dissociation. tRNA Asp split in the anticodon by T 1 ribonuclease gives no relaxation signal, indicating that the effects observed with intact tRNA were entirely due to anticodon interactions. No duplex formation was observed with other tRNAs having quasi self-complementary GNC anticodons (where N is C, A or G), such as E. coli tRNA Gly (GCC), E. coli tRNA Val (GAC) or E. coli tRNA Ala (GGC). This is compatible with the idea that, probably as in the crystal structure, a short double helix is formed in solution between the two GUC anticodons. Because of steric effects, such a complex formation would be hindered if a cytosine, adenine or guanine residue were located in the middle position of the anticodon. Escherichia coli tRNA Asp possessing a modified G residue, the Q base, at the first position of the anticodon, showed a weaker self-association than yeast tRNA Asp but its complex with E. coli tRNA Val was found to be only 1.5 times less stable than that between yeast tRNA Asp and E. coli tRNA Val. Temperature-jump experiments conducted under conditions mimicking those used for the crystallization of yeast tRNA Asp (in the presence of 1.6 m-ammonium sulphate and 3 m m-spermine) revealed an important stabilization of the yeast and E. coli tRNA Asp duplexes or of their complexes with E. coli tRNA Val. The effect is due exclusively to ammonium sulphate; it is entropy driven and its influence is reflected on the association rate constant; no influence on the dissociation rate constant was observed. For all tRNA-tRNA complexes, the melting temperature upon addition of ammonium sulphate was considerably increased. This study permits the definition of solution conditions in which tRNAs with appropriate anticodons exist mainly as anticodon-anticodon dimers.

Tetrahymena thermophila glutamine tRNA and its gene that corresponds to UAA termination codon.

Nucleotide sequence analysis of one of several tRNA genes cloned from Tetrahymena thermophila macronuclear DNA indicated that it corresponds to a tRNA species having TTA as anticodon. Subsequently, the tRNA species corresponding to that gene was isolated and its nucleotide sequence was determined by post-labeling techniques. The nucleotide sequence was found to be pG-G-U-U-C-C-A-U-A-m2G-U-A-psi-A-G-D-G-G-D- D-A-G-U-A-C-U-G-G-G-G-A-Cm-U-Um-U-A-i6A-A-psi-C-C-C-U-U-G-A-C- m5C-U-G-G-G-U-psi-C-G-m1A-A-U-C-C-C-A-G-U-G-G-G-A-C-C-U-C-C-AOH. This tRNA sequence exactly matched the DNA sequence of the corresponding tRNA gene. The first position of anticodon is 2'-O-methyluridine (Um), forming UmUA as the anticodon, which presumably recognizes the ochre termination codon UAA. This tRNA species is aminoacylated with glutamine by a Tetrahymena crude aminoacyl tRNA synthetase fraction, suggesting that ochre termination codon is used as a glutamine codon during cytoplasmic protein synthesis in Tetrahymena.

Nuclear magnetic resonance in biochemistry

Novel applications of nuclear magnetic resonance (NMR) spectroscopy in biochemistry are reviewed. The biological functions of peptides are correlated with the conformations of membrane-bound molecules rather than the conformations in aqueous solutions. The membrane-bound conformations may be elucidated by the analysis of transferred nuclear Overhauser effects. The conformations of nucleotide inhibitors as bound to ribonucleases may be analysed by nuclear Overhauser effect. Thus, ribonuclease T1 is found to have binding sites for guanine base and 3'-phosphate but not for 5'-phosphate group. From the 13C-NMR spectra of subtilisin inhibitors as labeled with [1-13C] Met and [15N]-Val, the inhibitor is found to form a Michaelis complex with subtilisin. From the proton NMR analyses of immunoglobin (IgG), the hinge region (indispensable for the activation of complement system) is found to be the most mobile. The uridine residue in the first position of anticodon of transfer ribonucleic acids (tRNA) are always modified after transcription. The conformational characteristics of the modified uridines have been elucidated by proton NMR analyses. The modifications of uridine in the first position of anticodon are now found to contribute to correct and efficient translations of codons, through the regulation of rigidity/flexibility of the anticodon moiety of tRNA.

Conformational aspects and biological functions of biomolecules

For studying the molecular mechanism of the biological function of transfer ribonucleic acid (tRNA, essential cell component for translation), NMR spin coupling constants, lanthanide-induced shifts and relaxation rates, and nuclear Overhauser effects have been analyzed of uridine phosphates and modified uridine derivatives in aqueous solution. On the basis of the conformational characteristics of uridine derivatives, the molecular mechanism of enhanced thermostability of extreme thermophile tRNA species and the biological significance of the two types of modifications of uridine in the first position of anticodon have been elucidated.

The yeast tRNATyr gene intron is essential for correct modification of its tRNA product.

Precise deletion of the intervening sequence of a yeast tRNATyr ochre suppressor gene (SUP6) significantly reduced its suppressor activity relative to that of the unaltered gene. This is probably the result of the absence of the pseudouridine modification, normally present at the middle anticodon position of mature suppressor tRNA, in tRNA synthesized in vivo from the deleted gene.

Changes in the amino acid code

The genetic code is characterized by a pattern arising from “wobble-pairing” between codons and anticodons, so that one nucleotide in the first anticodon position can pair with more than one nucleotide in the third position of a codon [1]. Earlier codes may have existed in which there were fewer anticodons than at present, so that these earlier codes contained fewer amino acids. The universal code was formerly thought to be the only currently existing code used by terrestrial species. It is now known that differences exist from the universal code in mitochondrial coding systems, and also that mitochondrial systems differ from each other. These findings lend support to the proposal that archetypal codes preceded the present universal code. Such archetypal codes may have had some resemblances to mitochondrial codes.

Sequences of halobacterial tRNAs and the paucity of U in the first position of their anticodons.

The sequence of three tRNAs from Halobacterium cutirubrum have been determined. The sequences of tRNAValGAC and tRNAValCAC differ by only one nucleotide which is in the 5' terminal anticodon position. These tRNAs as well as that of tRNAAlaCGC are compared to other known halobacterial tRNAs. An observed paucity (or absence) of U in the first anticodon position is unique to archaebacterial tRNAs and may be indicative of unusual decoding properties of these organisms.

Correct codon-anticoden base pairing at the 5'-anticodon position blocks covalent crosslinking between transfer ribonucleic acid and 16S RNA at the ribosomal P site

Correct codon-anticoden base pairing at the 5'-anticodon position blocks covalent crosslinking between transfer ribonucleic acid and 16S RNA at the ribosomal P site

A phenylalanine tRNA gene from Neurospora crassa: conservation of secondary structure involving an intervening sequence

We have isolated and sequenced a tRNAPhe gene from Neurospora crassa. Hybridization analyses suggest that trnaPhe is the only tRNA encoded on the cloned 5 kb DNA fragment. The tRNAPhe gene contains an intervening sequence 16 nucleotides in length located one nucleotide 3' to the anticodon position. The tRNAPhe coding region of Neurospora and yeast are 91% conserved, whereas their intervening sequences are only 50% identical. The pattern of sequence conservation is consistent with a proposed secondary structure for the tRNA precursor in which the anticodon is base paired with the middle of the intervening sequence and the splice points are located in adjacent single-stranded loops. The DNA sequence following the tRNAPhe coding region is similar to sequences following other genes transcribed by RNA polymerase III in that it is AT-rich and includes a tract of A residues in the coding strand. In contrast, the sequence preceding the Neurospora tRNAPhe coding region does not resemble sequences preceding other sequenced tRNA genes.