tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition.

In eubacteria, the biosynthesis of queuine, a modified base found in the wobble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway. One of the key enzymes in this pathway, tRNA-guanine transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1. Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the second position of its seven base anticodon loop. Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited structural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E. coli tRNA(Tyr) minihelix. The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants. Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU(+1)). The other analogs were not active. This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem. In E. coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33. U33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. We find that mutation of this base confers on TGT the ability to recognize U34G35U36, and suggests that loop conformation affects recognition. The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate.     

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.     

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.     

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.     

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.     

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.     

Loop stereochemistry and dynamics in transfer RNA

The stereochemistry and the dynamics of two loops of yeast tRNA-asp, the thymine loop and the anticodon loop, are compared in the hope of a better understanding of the relationships between loop sequence and loop topology. Both loops are seven residues long and both present sharp turns after the second residue, U33 and psi 55, stabilized by hydrogen bonds between N3-H of the pyrimidine and the phosphates of C36 and A58 and stacking interactions of the pyrimidine ring with the phosphates of U35 and A57, respectively. In the thymine loop, the two purines following C56, A57 and A58, open up to leave space for the intercalation of the first invariant guanine residue of the D-loop, while the two pyrimidine bases, which follow A58, turn away from the stacking pattern of the thymine arm and stack instead with the last base pair of the dihydrouridine arm A15-U48. In the anticodon loop, however, the bases G34 to C38 form an helical stack in continuity with the anticodon stem on the 3'-end. At the same time C36 forms Watson-Crick hydrogen bonds with G34 of a twofold symmetrically related molecule. The anticodon-anticodon base pairing interactions between symmetrically-related molecules are stabilized by stacking with the modified base G37 on both sides of the triplet. Some comparisons are made with the structure of yeast tRNA-phe and some implications about the structure of mitochondrial tRNAs are discussed.     

Recognition and positioning of mRNA in the ribosome by tRNAs with expanded anticodons

Mutant tRNAs containing an extra nucleotide in the anticodon loop are known to suppress +1 frameshift mutations, but in no case has the molecular mechanism been clarified. It has been proposed that the expanded anticodon pairs with a complementary mRNA sequence (the frameshift sequence) in the A site, and this quadruplet "codon-anticodon" helix is translocated to the P site to restore the correct reading frame. Here, we analyze the ability of tRNA analogs containing expanded anticodons to recognize and position mRNA in ribosomal complexes in vitro. In all cases tested, 8 nt anticodon loops position the 3' three-quarters of the frameshift sequence in the P site, indicating that the 5' bases of the expanded anticodon (nucleotides 33.5, 34, and 35) pair with mRNA in the P site. We also provide evidence that four base-pairs can form between the P-site tRNA and mRNA, and the fourth base-pair involves nucleotide 36 of the tRNA and lies toward (or in) the 30 S E site. In the A site, tRNA analogs with the expanded anticodon ACCG are able to recognize either CGG or GGU. These data imply a flexibility of the expanded anticodon in the A site. Recognition of the 5' three-quarters of the frameshift sequence in the A site and subsequent translocation of the expanded anticodon to the P site results in movement of mRNA by four nucleotides, explaining how these tRNAs can change the mRNA register in the ribosome to restore the correct reading frame.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.     

Non-bridging phosphate oxygen atoms within the tRNA anticodon stem-loop are essential for ribosomal A site binding and translocation

The conformation of the anticodon stem-loop of tRNAs required for correct decoding by the ribosome depends on intramolecular and intermolecular interactions that are independent of the tRNA nucleotide sequence. Non-bridging phosphate oxygen atoms have been shown to be critical for the structure and function of several RNAs. However, little is known about the role they play in ribosomal A site binding and translocation of tRNA to the P site. Here, we show that non-bridging phosphate oxygen atoms within the tRNA anticodon stem-loop at positions 33, 35, and 37 are important for A site binding. Those at positions 34 and 36 are not necessary for binding, but are essential for translocation. Our results correlate with structural data, indicating that position 34 interacts with the highly conserved 16S rRNA base G966 and position 36 interacts with the universally conserved tRNA base U33 during translocation to the P site.     

Yeast mitochondrial methionine initiator tRNA: characterization and nucleotide sequence.

Two methionine tRNAs from yeast mitochondria have been purified. The mitochondrial initiator tRNA has been identified by formylation using a mitochondrial enzyme extract. E. coli transformylase however, does not formylate the yeast mitochondrial initiator tRNA. The sequence was determined using both 32P-in vivo labeled and 32P-end labeled mt tRNAf(Met). This tRNA, unlike N. crassa mitochondrial tRNAf(Met), has two structural features typical of procaryotic initiator tRNAs: (i) it lacks a Watson-Crick base-pair at the end of the acceptor stem and (ii) has a T-psi-C-A sequence in loop IV. However, both yeast and N. crassa mitochondrial initiator tRNAs have a U11:A24 base-pair in the D-stem unlike procaryotic initiator tRNAs which have A11:U24. Interestingly, both mitochondrial initiator tRNAs, as well as bean chloroplast tRNAf(Met), have only two G:C pairs next to the anticodon loop, unlike any other initiator tRNA whatever its origin. In terms of overall sequence homology, yeast mitochondrial tRNA(Met)f differs from both procaryotic or eucaryotic initiator tRNAs, showing the highest homology with N. crassa mitochondrial initiator tRNA.     

Codon reading by tRNAAla with modified uridine in the wobble position

tRNAs reading four-codon families often have a modified uridine, cmo(5)U(34), at the wobble position of the anticodon. Here, we examine the effects on the decoding mechanism of a cmo(5)U modification in tRNA(1B)(Ala), anticodon C(36)G(35)cmo(5)U(34). tRNA(1B)(Ala) reads its cognate codons in a manner that is very similar to that of tRNA(Phe). As Ala codons are GC rich and Phe codons AU rich, this similarity suggests a uniform decoding mechanism that is independent of the GC content of the codon-anticodon duplex or the identity of the tRNA. The presence of cmo(5)U at the wobble position of tRNA(1B)(Ala) permits fairly efficient reading of non-Watson-Crick and nonwobble bases in the third codon position, e.g., the GCC codon. The ribosome accepts the C-cmo(5)U pair as an almost-correct base pair, unlike third-position mismatches, which lead to the incorporation of incorrect amino acids and are efficiently rejected.     

Interaction of tRNAs with the ribosome at the A and P sites.

In vitro transcribed tRNA(Phe) analogues from Escherichia coli containing up to four randomly distributed A, G, U or C phosphorothioated nucleotides were used to investigate contact patterns with the ribosome in the A and P sites. The tRNAs were biologically active. Molecular iodine (I2) can trigger a break in the sugar-phosphate backbone at phosphorothioated positions of the ribosomal bound tRNAs if contacts with ribosomal components do not prevent access of the iodine. Highly differentiated protection patterns were found which were strikingly different in the A and P sites, respectively. Strong protections accumulated in the T psi C loop and no protection was seen in the extra-arm region in both sites, whereas the phosphates in the anticodon loop are more strongly protected in the A site. Strong common protections in both the A and P sites were found neighbouring universally or semi-universally conserved bases in prominent regions of the tertiary structure of tRNAs: Y11, Y32, U33, psi55, C56, A58 and Y60. These bases are therefore candidates for 'identity elements' in ribosomal tRNA recognition. The data further indicate that tRNAs change their conformations upon binding to either ribosomal site.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Sequence and structure requirements for the biosynthesis of pseudouridine (psi 35) in plant pre-tRNA(Tyr).

All eukaryotic cytoplasmic tRNAs(Tyr) contain pseudouridine in the centre of the anticodon (psi 35). Recently, it has been shown that the formation of psi 35 is dependent on the presence of introns in tRNA(Tyr) genes. Furthermore, we have investigated the structural and sequence requirements for the biosynthesis of psi 35. A number of mutant genes were constructed by oligonucleotide-directed mutagenesis of a cloned Arabidopsis tRNA(Tyr) gene. Nucleotide exchanges were produced in the first and third positions of the anticodon and at positions adjacent to the anticodon. Moreover, insertion and deletion mutations were made in the anticodon stem and in the intron. The mutant genes were transcribed in HeLa cell extract and the pre-tRNAs(Tyr) were used for studying psi 35 biosynthesis in HeLa cell and wheat germ extracts. We have made the following observations about the specificity of plant and vertebrate psi 35 syntheses: (i) insertion or deletion of one base pair in the anticodon stem does not influence the efficiency and accuracy of the psi 35 synthase; (ii) the presence of U35 in a stable double-stranded region prevents its modification to psi 35; and (iii) the consensus sequence U33N34U35A36Pu37 in the anticodon loop is an absolute requirement for psi 35 synthesis. Thus, psi 35 synthases recognize both tRNA tertiary structure and specific sequences surrounding the nucleotide to be modified.