Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.     

Site-directed in vitro replacement of nucleosides in the anticodon loop of tRNA: application to the study of structural requirements for queuine insertase activity.

We have investigated the specificity of the enzymes Q-insertase and mannosyl-Q transferase that replace the guanosine at position 34 (wobble base) in the anticodon of several tRNAs by Q or mannosyl-Q derivatives. We have restructured in vitro the normal anticodon of yeast tRNA-Asp-GUC, yeast tRNAArgICG and yeast tRNALeuUAG. With yeast tRNA-Asp-GUC, we have replaced one or several nucleotides in the vicinity of G34 by one of the four canonical nucleotides or by pseudouridylic acid; we have also constructed a tRNAAsp with eight bases instead of seven in the anticodon loop. With yeast tRNAArgICG and yeast tRNALeuUAG, we have replaced their anticodon by the trinucleotide GUC, coding for aspartic acid. The chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and after 72 h the amount of Q34 and mannosyl-Q34 incorporated was measured. Our results show that the U33G34U35 sequence, within an anticodon loop of seven bases in chimerical yeast tRNA-Asp-GUC, tRNAArgGUC or tRNALeuGUC, is the main determinant for Q-insertase activity at position 34; the rest of the tRNA sequence has only a slight influence. For mannosyl-Q transferase, however, a much broader structural feature of the tRNA than just the U33G34U35 sequence is important for the efficiency of Q34 transformation into mannosyl-Q34.     

Accessible and inaccessible bases in yeast phenylalanine transfer RNA as studied by chemical modification.

The selective modification of cytidine, uridine, guanosine and dihydrouridine residues in ³²P-labelled yeast phenylalanine transfer RNA has been studied by the use of specific reagents.The selective modification of cytidine residues with the reagent methoxyamine is described. Of the six cytidines in the single-stranded regions of the cloverleaf formula, only two are completely reactive, C74 and C75 at the 3′-terminus. Cm32 in the anticodon loop is reactive to only a small extent.The selective modifications of uridine and guanosine residues with 1-cyclohexyl 3-[2-morpholino(4)-ethyl] carbodiimide methotosylate, is described. The reagent is also shown to be reactive with dihydrouridine. In the single-stranded regions of the secondary structure of yeast phenylalanine transfer RNA there are 16 base residues which this reagent could be specific for. However, only G20, Gm34 and U47 are extensively modified, whilst U33 and D16 are partially modified. G18 is modified to a very small extent.The results obtained in this study are also in good agreement with previous chemical modification studied by other workers, carried out on unlabelled yeast phenylalanine transfer RNA using different reagents to the ones described here.The pattern of chemical modification is compared with the three-dimensional structure obtained by an X-ray crystallographic analysis of the same tRNA species. The correlation between exposed regions of the model and the regions of chemical reactivity are everywhere consistent.     

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.     

Effect of threonylcarbamoyl modification (t6A) in yeast tRNA Arg III on codon-anticodon and anticodon-anticodon interactions. A thermodynamic and kinetic evaluation

The effect of N-[9-(beta-D-ribofuranosyl) purin-6-ylcarbamoyl]threonine (t6A) adjacent to anticodon U-C-U of yeast tRNA Arg III (where U is a modified U), compared to its unmodified adenosine counterpart, has been evaluated by three independent methods: (a) the polynucleotide-directed binding of tRNA on ribosomes, (b) the ribosome-free trinucleotide binding to the anticodon, (c) the anticodon-anticodon binding test. The results obtained by these three methods indicate a small but significant stabilization effect of t6A on the binding of yeast tRNA Arg III with (a) poly(A,G) in the presence of Escherichia coli ribosomes, (b) free A-G-A triplet, and (c) E. coli tRNA Ser V (anticodon G-G-A). We therefore conclude that the stabilization effect of t6A occurs on U x A and U x G base pairs adjacent to the 5' side of the modified nucleoside, most probably by stacking.     

Enzymatic replacement in vitro of the first anticodon base of yeast tRNAAsp: application to the study of tRNA maturation in vivo, after microinjection into frog oocytes.

A combination of several enzymes, RNase-T1, nuclease S1, T4-polynucleotide kinase and T4-RNA ligase were used to prepare and modify different fragments of yeast tRNAAsp (normal anticodon G U C). This allowed us to reconstitute, in vitro, a chimeric tRNA that has any of the four bases G, A, U or C, as the first anticodon nucleotide, labelled with (32p) in its 3' position. Such reconstituted (32p) labelled yeast tRNAAsp were microinjected into the cytoplasm or the nucleus of the frog oocyte and checked for their stability as well as for their potential to work as a substrate for the maturation (modifying) enzymes under in vivo conditions. Our results indicate that the chimeric yeast tRNAsAsp were quite stable inside the frog oocyte. Also, the G34 was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q34; U34 into mcm5s2U and mcm5U. In contrast, C34 and A34 were not transformed at all neither in the cytoplasm nor in the nucleus of the frog oocyte. The above procedure constitutes a new approach in order to detect the presence of a given modifying enzyme inside the frog oocyte; also it provides informations about its cellular location and possibility about its specificity of interaction with foreign tRNA.     

Enzymatic formation of queuosine and of glycosyl queuosine in yeast tRNAs microinjected into Xenopus laevis oocytes. The effect of the anticodon loop sequence

The eukaryotic tRNA-guanine transglycosylases (queuine insertases) catalyse an exchange of guanine for queuine in position 34, the wobble nucleoside, of tRNAs having a GUN anticodon where N (position 36) stands for A, U, C or G. In tRNAAsp (anticodon QUC) and tRNATyr (anticodon Q psi A) from certain eukaryotic cells, the nucleoside Q-34 is further hypermodified into a glycosylated derivative by tRNA-queuine glycosyltransferase. In order to gain insight into the influence of the nucleosides in position 36, 37 and 38 of an anticodon loop on the potential of a tRNA to become a substrate for the two modifying enzymes, we have constructed several variants of yeast tRNAs in which the normal anticodon has been replaced by one of the synthetic anticodons GUA, GUC, GUG or GUU. In yeast tRNAAsp, the nucleosides 37 (m1G) and 38(C) have also been replaced by an adenosine. These reconstructed chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and tested for their ability to react with the oocyte maturation enzymes. Our results indicate that the nucleosides in positions 36, 37 and 38 influence the efficiencies of conversion of G-34 to Q-34 and of Q-34 to glycosyl Q-34; the importance of their effects are much more pronounced on the glycosylation of Q-34 than on the insertion of queuine. The effect of the nucleoside in position 37 is of particular importance in the case of yeast tRNAAsp: the replacement of the naturally occurring m1G-37 by an unmodified adenosine (as it is in X. laevis tRNAAsp), considerably increases the yield of the glycosylation reaction catalysed by the X. laevis tRNA-queuine glycosyltransferase.     

Intron-dependent formation of pseudouridines in the anticodon of Saccharomyces cerevisiae minor tRNA(Ile).

We have isolated and sequenced the minor species of tRNA(Ile) from Saccharomyces cerevisiae. This tRNA contains two unusual pseudouridines (psi s) in the first and third positions of the anticodon. As shown earlier by others, this tRNA derives from two genes having an identical 60 nt intron. We used in vitro procedures to study the structural requirements for the conversion of the anticodon uridines to psi 34 and psi 36. We show here that psi 34/psi 36 modifications require the presence of the pre-tRNA(Ile) intron but are not dependent upon the particular base at any single position of the anticodon. The conversion of U34 to psi 34 occurs independently from psi 36 synthesis and vice versa. However, psi 34 is not formed when the middle and the third anticodon bases of pre-tRNA(Ile) are both substituted to yield ochre anticodon UUA. This ochre pre-tRNA(Ile) mutant has the central anticodon uridine modified to psi 35 as is the case for S.cerevisiae SUP6 tyrosine-inserting ochre suppressor tRNA. In contrast, neither the first nor the third anticodon pseudouridine is formed, when the ochre (UUA) anticodon in the pre-tRNA(Tyr) is substituted with the isoleucine UAU anticodon. A synthetic mini-substrate consisting of the anticodon stem and loop and the wild-type intron of pre-tRNA(Ile) is sufficient to fully modify the anticodon U34 and U36 into psi s. This is the first example of the tRNA intron sequence, rather than the whole tRNA or pre-tRNA domain, being the main determinant of nucleoside modification.     

C to U editing stimulates A to I editing in the anticodon loop of a cytoplasmic threonyl tRNA in Trypanosoma brucei

Editing of tRNAs is widespread in nature and either changes the decoding properties or restores the folding of a tRNA. Unlike the phylogenetically disperse adenosine (A) to inosine (I) editing, cytosine (C) to uridine (U) editing has only been previously described in organellar tRNAs. We have shown that cytoplasmic tRNA(Thr)(AGU) undergoes two distinct editing events in the anticodon loop: C to U and A to I. In vivo, every inosine-containing tRNA(Thr) is also C to U edited at position 32. In vitro, C to U editing stimulates conversion of A to I at the wobble base. Although the in vivo and in vitro requirements differ, in both cases, the C to U change plays a key role in A to I editing. Due to an unusual abundance of A34-containing tRNAs, our results also suggest that the unedited and edited tRNAs are functional, each dedicated to decoding a specific threonine codon. C to U editing of cytoplasmic tRNA expands the editing repertoire in eukaryotic cells, and when coupled to A to I changes, leads to an interrelation between editing sites.     

Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.     

The structure of yeast tRNA(Asp). A model for tRNA interacting with messenger RNA

The anticodon of yeast tRNA(Asp), GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA(Asp) molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA(Phe). In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA(Asp) T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA(Phe). This variation is a consequence of the anticodon-anticodon base pairing which rigidifies the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA(Asp) substantiate such a correlation.     

Mitochondria-specific RNA-modifying enzymes responsible for the biosynthesis of the wobble base in mitochondrial tRNAs. Implications for the molecular pathogenesis of human mitochondrial diseases

Human mitochondrial (mt) tRNA(Lys) has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (taum5s2U), at its anticodon wobble position. We previously found that the mt tRNA(Lys), carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the taum5s2U modification. Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity. Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed. Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding. Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF.     

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

Summary The 20 naturally occurring amino acids are characterized by 20 variables: pK_{NH 2}, pK_COOH, pI, molecular weight, substituent van der Waals volume, seven¹H and¹³C 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.     

Modification of tRNA 1 Val from yeast with monoperphthalic acid]

A method is proposed for analysis of natural and chemically modified polynucleotides which consists in enzymatic conversion of the polymer or oligomer into nucleosides followed by cation-exchange chromotography on the microcolumns. By using the method developed it was shown that after treatment of the yeast tRNAVal and tRNAPhe with monoperphthalic acid N-oxides of adenosine and cytidine were formed. Poly (U, G) was not modified at a measurable extent whereas GMP was decomposed. In tRNAVal (yeast)the adenosines and cytosines of the anticodon loop and 3'-end are most reactive; it is the case for the C17 of the diHU-loop as well. These data are in agreement with the results obtained for tRNA modification with other reagents and for limited enzymatic hydrolysis of the tRNAVal. The limitations of the reaction of the monoperphthalate with nucleic acids are briefly discussed.     

Kluyveromyces lactis gamma-toxin, a ribonuclease that recognizes the anticodon stem loop of tRNA

Kluyveromyces lactis gamma-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae [see text] between position 34 and position 35. All three substrate tRNAs carry a 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) residue at position 34 (wobble position) of which the mcm(5) group is required for efficient cleavage. However, the different cleavage efficiencies of mcm(5)s(2)U(34)-containing tRNAs suggest that additional features of these tRNAs affect cleavage. In the present study, we show that a stable anticodon stem and the anticodon loop are the minimal requirements for cleavage by gamma-toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate [see text] is cleaved at the same position as the natural substrate. In [see text], the nucleotides U(34)U(35)C(36)A(37)C(38) are required for optimal gamma-toxin cleavage, whereas a purine at position 32 or a G in position 33 dramatically reduces the cleavage of the ASL. Comparing modified and partially modified forms of E. coli and yeast [see text] reinforced the strong stimulatory effects of the mcm(5) group, revealed a weak positive effect of the s(2) group and a negative effect of the bacterial 5-methylaminomethyl (mnm(5)) group. The data underscore the high specificity of this yeast tRNA toxin.