Crystallization and preliminary X-ray analysis of the archaeosine tRNA-guanine transglycosylase from Pyrococcus horikoshii.

The archaeosine tRNA-guanine transglycosylase from the hyperthermophilic archaeon Pyrococcus horikoshii was crystallized and preliminary X-ray characterization was performed. Single crystals were grown by the hanging-drop vapour-diffusion method, using sodium/potassium phosphate and sodium acetate as precipitants. The space group is P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 99.28 (14), c = 363.74 (56) A. The cryocooled crystals diffracted X-rays beyond 2.2 A resolution using synchrotron radiation from station BL44XU at SPring-8 (Harima). Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the MAD phasing method.

tRNA recognition of tRNA-guanine transglycosylase from a hyperthermophilic archaeon, Pyrococcus horikoshii.

In the biosynthesis of archaeosine, archaeal tRNA-guanine transglycosylase (TGT) catalyzes the replacement of guanine at position 15 in the D loop of most tRNAs by a free precursor base. We examined the tRNA recognition of TGT from a hyperthermophilic archaeon, Pyrococcus horikoshii. Mutational studies using variant tRNA(Val) transcripts revealed that both guanine and its location (position 15) were strictly recognized by TGT without any other sequence-specific requirements. It appeared that neither the global L-shaped structure of a tRNA nor the local conformation of the D loop contributed to recognition by TGT. A minihelix composed of the acceptor stem and D arm of tRNA(Val), designed as a potential minimal substrate, failed to serve as a substrate for TGT. Only a minihelix with mismatched nucleotides at the junction between the two domains served as a good substrate, suggesting that mismatched nucleotides in the helix provide the specific information that allows TGT to recognize the guanine in the D loop. Our findings indicate that the tRNA recognition requirements of P. horikoshii TGT are sufficiently limited and specific to allow the enzyme to recognize efficiently any tRNA species whose structure is not fully stabilized in an extremely high temperature environment.

Three of four pseudoknots in tmRNA are interchangeable and are substitutable with single-stranded RNAs.

A novel translation, trans-translation, is facilitated by a highly structured RNA molecule, tmRNA. This molecule has two structural domains, a tRNA domain and an mRNA domain, the latter including four pseudoknot structures (PK1 to PK4). Here, we show that replacement of each of these pseudoknots, except PK1, in Escherichia coli tmRNA with a single stranded RNA did not seriously affect the functions as an alanine tRNA and as an mRNA. Furthermore, these three pseudoknots were interchangeable with only small losses of the two functions. These findings suggest that neither PK2, PK3 nor PK4 interacts in a functional manner with ribosome during the trans-translation process. Together with an earlier study showing the significance of PK1, it is concluded that among the four pseudoknots, PK1 is the most functional.

An NMR and mutational analysis of an RNA pseudoknot of Escherichia coli tmRNA involved in trans-translation.

Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed trans -translation. According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350-400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes. A mutational analysis using a number of Escherichia coli tmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in trans -translation. Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudoknot structure in the presence of Mg(2+). Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems. Structural instability of the pseudoknot structure, even in the presence of Mg(2+), was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activities indicate that trans -translation requires the pseudoknot structure stabilized by Mg(2+)and specific residues G61 and G62 in loop 3.

Amino acid acceptor identity switch of Escherichia coli tmRNA from alanine to histidine in vitro.

According to a trans -translation model, tmRNA facilitates the resumption of translation that has been stalled on the ribosome with the 3' end of a terminator-less mRNA, to produce a chimera polypeptide of the nascent peptide and the tmRNA-encoding 11 amino acid-tag. The first alanine residue of the tag-sequence is encoded neither by mRNA nor by tmRNA. This alanine is a key molecule for this model, in which it is derived from the alanine moiety aminoacylated to tmRNA. This is supported only by the observation that a point mutation at the third base-pair position of the acceptor stem of Escherichia coli tmRNA that deprives it of its aminoacylation ability causes abolishment of tag-peptide synthesis in vitro. Here, we made an E. coli tmRNA mutant with a completely switched amino acid acceptor identity from alanine to histidine by transplanting the upper half of the acceptor stem of tRNAHis. This histidine acceptor tmRNA mutant still retained an ability of tag-specific amino acid incorporation into the polypeptide in an in vitro poly(U)-dependent tag-peptide synthesis system, with an altered amino acid composition. Histidine, which is not a constituent of the original tag-peptide, was incorporated into the mutant-directed tag. The molar ratio of amino acids incorporated is consistent with that in the tag-sequence with the only expected change being the first amino acid from alanine to histidine. These results indicate that the first alanine residue of the tag-peptide is actually derived from that aminoacylated to tmRNA and is substitutable by other amino acids during the trans -translation processes.

Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E. coli tmRNA.

Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-translation reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 11 amino acid residue tag to the truncated protein that is a signal for degradation and in recycling of the stalled ribosome. A tmRNA secondary structure model with a partial tRNA-like structure and several pseudoknots was recently proposed. This report describes an extensive mutational analysis of one predicted pseudoknot (PK1) located upstream of the E. coli tmRNA tag-encoded sequence. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all the engineered RNA variants. To characterize structure-function relationships for the tmRNA mutants, their solution conformations were investigated by using structural probes and by measuring the temperature dependence of their UV absorbance. This analysis strongly supports the presence of a pseudoknot in E. coli tmRNA, and its involvement in trans-translation. Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations. Mutations of the second stem of the pseudoknot also effect both functions. The nucleotide stretch between the two stems (loop 2) is required for efficient trans-translation, and nucleotides at positions 61 and 62 must be guanine residues. The probing data suggest the presence of magnesium ion(s) interacting with loop 2. The loops crossing the minor and major grooves can be mutated without significant effects on tmRNA function. Nucleotide insertion or deletion between the pseudoknot and the coding sequence do not change the mRNA frame of the tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation.

In vitro selection of RNAs aminoacylated by Escherichia coli leucyl-tRNA synthetase.

To investigate systematically the RNA sequences necessary for aminoacylation by Escherichia coli leucyl-tRNA synthetase, RNAs with leucylation activity were isolated by in vitro selection from a library of tRNALeu variants possessing randomized sequences in the D-loop, the variable arm, and the T-loop. After two rounds of selection, most of the selected variants showed the following features: (1) the tertiary interaction between nucleotides at positions 15 and 48 was A15-U48; (2) the continuous G18G19 sequence, which is invariant in canonical tRNAs, appeared at the fixed position in the D-loop; and (3) the nucleotide at position 20a in the D-loop was A. These selected nucleotides and their positions, concentrating on the hinge region of tRNA, were identical to those of native tRNALeu. In contrast, although the long variable arm is the most characteristic of the tRNALeu structure, the primary and secondary structures were not correlated with the leucylation activity. These findings indicate that A15-U48, A20a, and G18G19 located at specific positions are involved in the tertiary folding of leucine-accepting tRNA molecules. With increases in the selection cycle, the D-loop sequence and the secondary structure of the variable arm became similar to those of tRNALeu, suggesting that tRNALeu represents an optimized RNA sequence for leucylation.

Recognition of tRNA(Gly) by three widely diverged glycyl-tRNA synthetases.

Glycyl-tRNA synthetase (GlyRS) is an unusual aminoacyl-tRNA synthetase because it varies in its quarternary structure between organisms; Escherichia coli GlyRS is an alpha2beta2 tetramer, whereas those of Thermus thermophilus and yeast are alpha2 dimers. In contrast, the tRNA(Gly) sequence is virtually identical in E. coli and T. thermophilus but very different in yeast. In this study, we examined the molecular recognition of tRNA(Gly) by three widely diverged GlyRSs using in vitro tRNA transcripts. Mutation studies showed that the discriminator base at position 73, the second base-pair, C2 x G71, in the acceptor stem, and the anticodon nucleotides, C35 and C36, contribute to the specific aminoacylation of all three GlyRSs, the discriminator base differing between prokaryotes (U73) and eukaryotes (A73). However, we found differences between yeast and two bacteria around the second base-pair in the acceptor stem. The first base-pair, G1 x C72, is important for glycylation in E. coli and T. thermophilus, whereas the third base-pair, G3 x C70, is important for glycylation in yeast. These findings indicate that despite such large differences of the two prokaryotic GlyRSs, tRNA(Gly) identity has been essentially conserved in prokaryotes, and that there are also differences in the acceptor stem recognition between prokaryotes and yeast. The clear separation between prokaryotes and yeast is retained in the identity element location, whereas the apparent diversity of the two prokaryotic enzymes does not reflect on the tRNA recognition.

Recognition of tRNA(Gly) by three widely diverged glycyl-tRNA synthetases: evolution of tRNA recognition.

Glycyl-tRNA synthetase (GlyRS) is an unusual aminoacyl-tRNA synthetase because it varies in its quarternary structure between organisms; Escherichia coli GlyRS is an alpha 2 beta 2 tetramer, whereas those of Thermus thermophilus and yeast are alpha 2 dimers. In contrast, the tRNA(Gly) sequence is virtually identical in E. coli and T. thermophilus but very different in yeast. In this study, we examined the molecular recognition of tRNA(Gly) by three widely diverged GlyRSs using in vitro tRNA transcripts. The results obtained in the mutation studies indicate that despite such large differences of the two prokaryotic GlyRSs, tRNA(Gly) identity has been essentially conserved in prokaryotes, and that there are also differences in the acceptor stem recognition between prokaryotes and yeast. The clear separation between prokaryotes and yeast is retained in the identity element location, whereas the apparent diversity of the two prokaryotic enzymes does not reflect on the tRNA recognition.

Escherichia coli tmRNA (10Sa RNA) in trans-translation.

Here we show that Escherichia coli tmRNA (10Sa RNA) has a dual function both as an mRNA and as a tRNA in vitro. The function as a tRNA is prerequisite for the function as an mRNA. These observations strongly support the trans-translation hypothesis.

Identity elements of Thermus thermophilus tRNA(Thr).

In this study, we identified nucleotides that specify aminoacylation of tRNA(Thr) by Thermus thermophilus threonyl-tRNA synthetase (ThrRS) using in vitro transcripts. Mutation studies showed that the first base pair in the acceptor stem as well as the second and third positions of the anticodon are major identity elements of T. thermophilus tRNA(Thr), which are essentially the same as those of Escherichia coli tRNA(Thr). The discriminator base, U73, also contributed to the specific aminoacylation, but not the second base pair in the acceptor stem. These findings are in contrast to E. coli tRNA(Thr), where the second base pair is required for threonylation, with the discriminator base, A73, playing no roles. In addition, among several mutations at the third base pair in the acceptor stem, only the G3-U70 mutant was a poor substrate for ThrRS, suggesting that the G3-U70 wobble pair, which is the identity determinant of tRNA(Ala), acts as a negative element for ThrRS. Similar results were obtained in E. coli and yeast. Thus, this manner of rejection of tRNA(Ala) is also likely to have been retained in the threonine system throughout evolution.

Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase.

Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase were examined using in vitro transcripts. By mutation studies, a marked decrease in aminoacylation with threonine showed that the first base pair in the acceptor stem and the second and third positions of the anticodon are major identity elements of tRNA(Thr), which are essentially the same as those of Escherichia coli tRNA(Thr). Base substitution of the discriminator base, A73, by G73 or C73 impaired the threonine accepting activity, but not that by U73, suggesting that this position contributes to discrimination from other tRNAs possessing G73 or C73. No effects on aminoacylation were observed with substitutions at the second base pair in the acceptor stem. These are in contrast to E.coli tRNA(Thr) where the second base pair is required for the specific aminoacylation, with the discriminator base playing no roles. Of several mutations at the third base pair in the acceptor stem, only the G3-U70 mutation impaired the activity, suggesting that the G3-U70 wobble pair, the identity determinant of tRNAAla, acts as a negative element for threonyl-tRNA synthetase. These findings indicate that while the first base pair in the acceptor stem and the anticodon nucleotides have been retained as major recognition sites between S. cerevisiae and E.coli tRNA(Thr), the mechanism by which the synthetase recognizes the vicinity of the top of the acceptor stem seems to have diverged with the species.

Identity elements of Saccharomyces cerevisiae tRNA(His).

Recognition of tRNA(His) by Saccharomyces cerevisiae histidyl-tRNA synthetase was studied using in vitro transcripts. Histidine tRNA is unique in possessing an extra nucleotide, G-1, at the 5' end. Mutation studies indicate that this irregular secondary structure at the end of the acceptor stem is important for aminoacylation with histidine, while the requirement of either base of this extra base pair is smaller than that in Escherichia coli. The anticodon was also found to be required for histidylation. The regions involved in histidylation are essentially the same as those in E.coli, whereas the proportion of the contributions of the two portions distant from each other, the anticodon and the end of the acceptor stem, makes a substantial difference between the two systems.

Similarities and differences in tRNA identity between Escherichia coli and Saccharomyces cerevisiae: evolutionary conservation and divergence.

Identity elements, which allow correct recognition of tRNAs by their cognate aminoacyl-tRNA synthetase, have been well elucidated in Escherichia coli to begin to see a pattern for tRNA recognition. We examined the identity elements of several tRNA species from Saccharomyces cerevisiae and Thermus thermophilus using in vitro transcripts. Comparison of identity elements among different organisms indicates not only conservation but also evolutionary divergence of tRNA recognition.

Role of the CCA terminal sequence of tRNA(Val) in aminoacylation with valyl-tRNA synthetase.

All known tRNAs have a universal CCA sequence at the 3'-terminal. To study the role of this terminal sequence in the aminoacylation process, base substitutions were introduced into a transcript of Escherichia coli valine tRNA and the effects on the aminoacylation activity with valyl-tRNA synthetase were evaluated. Substitution of the terminal adenosine residue at position 76 by C or U caused a 5-7-fold decrease of valine charging activity in Vmax/Km, while substitution by G resulted in about a 300-fold decrease. In addition, these mutations gave rise to an appreciable level of misaminoacylation with threonine. ATP hydrolysis activity during threonylation was lower in the terminal adenosine mutants than in the wild-type. Mutations introduced at positions 75 and 74 also caused threonylation instead of reducing valylation, albeit to a much smaller extent. These results indicate that the CCA sequence, especially the base portion of the terminal adenosine residue, plays an important role not only in amino-acylation efficiency with valine but also in preventing misaminoacylation by hydrolyzing misactivated threonyl-tRNA(Val).

Escherichia coli seryl-tRNA synthetase recognizes tRNA(Ser) by its characteristic tertiary structure.

To investigate the sequence requirements of Escherichia coli tRNA(Ser) for recognition by seryl-tRNA synthetase, various mutants of unmodified tRNA(Ser) were constructed. Substitution of G2.C71 by C2.G71, but not by A2.U71 or U2.A71, impaired the serine-accepting activity, indicating that this position is not involved in recognition by seryl-tRNA synthetase, but contributes to discrimination from other tRNAs processing C2.G71 such as tRNA(Leu). Other nucleotides characteristic of tRNA(Ser), including the discriminator base, were not involved in recognition by seryl-tRNA synthetase. The anticodon was not involved, as suggested by its sequence variety within the isoacceptors. The long variable arm composed of over ten nucleotides, which is a characteristic feature of tRNA(Ser) together with tRNA(Leu) and tRNA(Tyr), was stem-length-specifically, but not sequence-specifically, important for recognition. In order to introduce a sufficient serine-accepting activity to a tRNA(1LEU) transcript in vitro, besides the change from C2.G71 to G2.C71, the following elements had to be changed to those characteristic of tRNA(Ser): the sequence in the D-loop, the stem pairing pattern of the variable arm, the tertiary base-pair 15.48 and the nucleotide at position 59 in the T psi C-loop. None of the nucleotides at these changed positions was involved in base-specific recognition, indicating that seryl-tRNA synthetase selectively recognizes tRNA(Ser) on the basis of its characteristic tertiary structure rather than the nucleotides specific to tRNA(Ser).

Discrimination among E. coli tRNAs with a long variable arm.

In E. coli, tRNA(Ser), tRNA(Leu) and tRNA(Tyr) have a long variable arm composed of more than ten nucleotides (class II tRNAs). In order to study how leucyl- and seryl-tRNA synthetase discriminate their cognate tRNA isoacceptors from the other class II tRNAs, kinetic parameters of various mutated class II tRNA transcripts with leucyl- and seryl-tRNA synthetase were determined. Leucyl-tRNA synthetase recognizes A73 and A14 or its vicinity. Seryl-tRNA synthetase recognizes the long variable arm base-nonspecifically. C2-G71 in the acceptor stem functions as a negative identity element against seryl-tRNA synthetase. Difference in the tertiary structure among class II tRNA molecules plays a crucial role in discrimination by these two synthetases.

Escherichia coli tRNA(Asp) recognition mechanism differing from that of the yeast system.

Various tRNA transcripts were constructed to study the identity elements of Escherichia coli tRNA(Asp). Base substitutions from G34 to U34 at the first position of the anticodon, and from U35 to A35 at the second, severely impaired the aspartate charging activity. The activity was also decreased, but in a more moderate fashion, by base changes at G2-C71, C36 and C38. Identity nucleotides of tRNA(Asp) are distributed in a different fashion between E. coli and yeast, which occur at the second base pair of the acceptor stem, G10-U25 base pair in the D-stem and 3' half of the anticodon loop.

In vitro study of E. coli tRNA identity elements.

Various tRNA transcripts were constructed to study the identity elements of E. coli tRNAs (Arg, Lys, Ala, Trp, Thr, Gly, Ser, Asn, Cys, His). Anticodon are involved in the identity elements in these tRNA species except the case of tRNA(Ala) and tRNA(Ser). Especially, the second and third positions of the anticodon are the recognition sites of E. coli tRNA(Arg), tRNA(Lys) and tRNA(Thr) for their cognate aminoacyl-tRNA synthetases. Discriminator base is an identity determinant of the above examined tRNAs except tRNA(Thr) and tRNA(Ser). In some cases, acceptor stem (Thr, Gly, His) and variable pocket (Arg, Ala) are considered to be the recognition elements.

Identity determinants of E. coli tRNAs.

Identity determinants of E. coli tRNA(Val) and three class II tRNAs, tRNA(Ser), tRNA(Tyr) and tRNA(Leu), are studied by using various variants of tRNA transcripts. Anticodon, discriminator base and acceptor stem are involved in the identity elements for tRNA(Val). Discrimination among class II tRNAs are considered to be dependent on the bases at positions 2, 71 and 73 as well as their different tertiary structures including the long variable arm.