Circular dichroism analyses of tRNA X protein interactions

The interactions of tRNA species with aminoacyl-tRNA synthetases and polypeptide chain elongation factor Tu from Thermus thermophilus HB8 were studied by the analyses mainly of the circular dichroism band of 2-thioribothymidine in position 54 of T. thermophilus tRNA species.     

Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase

Thermus thermophilus possesses two aspartyl-tRNA synthetases (AspRSs), AspRS1 and AspRS2, encoded by distinct genes. Alignment of the protein sequences with AspRSs of other origins reveals that AspRS1 possesses the structural features of eubacterial AspRSs, whereas AspRS2 is structurally related to the archaebacterial AspRSs. The structural dissimilarity between the two thermophilic AspRSs is correlated with functional divergences. AspRS1 aspartylates tRNA(Asp) whereas AspRS2 aspartylates tRNA(Asp), and tRNA(Asn) with similar efficiencies. Since Asp bound on tRNA(Asn) is converted into Asn by a tRNA-dependent aspartate amidotransferase, AspRS2 is involved in Asn-tRNA(Asn) formation. These properties relate functionally AspRS2 to archaebacterial AspRSs. The structural basis of the dual specificity of T. thermophilus tRNA(Asn) was investigated by comparing its sequence with those of tRNA(Asp) and tRNA(Asn) of strict specificity. It is shown that the thermophilic tRNA(Asn) contains the elements defining asparagine identity in Escherichia coli, part of which being also the major elements of aspartate identity, whereas minor elements of this identity are missing. The structural context that permits expression of aspartate and asparagine identities by tRNA(Asn) and how AspRS2 accommodates tRNA(Asp) and tRNA(Asn) will be discussed. This work establishes a distinct structure-function relationship of eubacterial and archaebacterial AspRSs. The structural and functional properties of the two thermophilic AspRSs will be discussed in the context of the modern and primitive pathways of tRNA aspartylation and asparaginylation and related to the phylogenetic connexion of T. thermophilus to eubacteria and archaebacteria.     

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N²-methylguanosine at position 6 in tRNA

N(2)-methylguanosine (m(2)G) is found at position 6 in the acceptor stem of Thermus thermophilus tRNA(Phe). In this article, we describe the cloning, expression, and characterization of the T. thermophilus HB27 methyltransferase (MTase) encoded by the TTC1157 open reading frame that catalyzes the formation of this modified nucleoside. S-adenosyl-L-methionine is used as donor of the methyl group. The enzyme behaves as a monomer in solution. It contains an N-terminal THUMP domain predicted to bind RNA and contains a C-terminal Rossmann-fold methyltransferase (RFM) domain predicted to be responsible for catalysis. We propose to rename the TTC1157 gene trmN and the corresponding protein TrmN, according to the bacterial nomenclature of tRNA methyltransferases. Inactivation of the trmN gene in the T. thermophilus HB27 chromosome led to a total absence of m(2)G in tRNA but did not affect cell growth or the formation of other modified nucleosides in tRNA(Phe). Archaeal homologs of TrmN were identified and characterized. These proteins catalyze the same reaction as TrmN from T. thermophilus. Individual THUMP and RFM domains of PF1002 from Pyrococcus furiosus were produced. These separate domains were inactive and did not bind tRNA, reinforcing the idea that the THUMP domain acts in concert with the catalytic domain to target a particular position of the tRNA molecule.     

Aspartyl-tRNA synthetase requires a conserved proline in the anticodon-binding loop for tRNA(Asn) recognition in vivo

Most prokaryotes require Asp-tRNA(Asn) for the synthesis of Asn-tRNA(Asn). This misacylated tRNA species is synthesized by a non-discriminating aspartyl-tRNA synthetase (AspRS) that acylates both tRNA(Asp) and tRNA(Asn) with aspartate. In contrast, a discriminating AspRS forms only Asp-tRNA(Asp). Here we show that a conserved proline (position 77) in the L1 loop of the non-discriminating Deinococcus radiodurans AspRS2 is required for tRNA(Asn) recognition in vivo. Escherichia coli trpA34 was transformed with DNA from a library of D. radiodurans aspS2 genes with a randomized codon 77 and then subjected to in vivo selection for Asp-tRNA(Asn) formation by growth in minimal medium. Only proline codons were found at position 77 in the aspS2 genes isolated from 21 of the resulting viable colonies. However, when the aspS temperature-sensitive E. coli strain CS89 was transformed with the same DNA library and then screened for Asp-tRNA(Asp) formation in vivo by growth at the non-permissive temperature, codons for seven other amino acids besides proline were identified at position 77 in the isolates examined. Thus, replacement of proline 77 by cysteine, isoleucine, leucine, lysine, phenylalanine, serine, or valine resulted in mutant D. radiodurans AspRS2 enzymes still capable of forming Asp-tRNA(Asp) but unable to recognize tRNA(Asn). This strongly suggests that proline 77 is responsible for the non-discriminatory tRNA recognition properties of this enzyme.     

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.     

Nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8.

The nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8, was determined by a combination of classical methods using unlabeled samples to determine the sequences of the oligonucleotides of RNase T1 and RNase A digests and a rapid sequencing gel technique using 5'-32P labeled samples to determine overlapping sequences. Formylmethionine tRNA from T. thermophilus is composed of two species, tRNAf1Met and tRNAf2Met. Their nucleotide sequences are almost identical, and are also almost identical with that of E. coli tRNAfMet, except for slight modifications and replacements. Both species have modifications at three points which do not exist in E. coli tRNAfMet: 2'-O-methylation at G19, N-1-methylation at A59 and 2-thiolation at T55. Moreover U51 in E. coli tRNAfMet is replaced by C51 in both species, so that a G-C pair is formed between this C51 and G65. tRNAf2Met has a reversed G-C pair at positions 52 and 64 compared with those in tRNAf1Met and E. coli tRNAfMet. Other regions are mostly the same as those in all prokaryotic initiator tRNAs so far reported. The thermostability of these thermophile initiator tRNAs is discussed in relation to their unique modifications.     

Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.     

The RNA-binding PUA domain of archaeal tRNA-guanine transglycosylase is not required for archaeosine formation

Bacterial tRNA-guanine transglycosylase (TGT) replaces the G in position 34 of tRNA with preQ(1), the precursor to the modified nucleoside queuosine. Archaeal TGT, in contrast, substitutes preQ(0) for the G in position 15 of tRNA as the first step in archaeosine formation. The archaeal enzyme is about 60% larger than the bacterial protein; a carboxyl-terminal extension of 230 amino acids contains the PUA domain known to contact the four 3'-terminal nucleotides of tRNA. Here we show that the C-terminal extension of the enzyme is not required for the selection of G15 as the site of base exchange; truncated forms of Pyrococcus furiosus TGT retain their specificity for guanine exchange at position 15. Deletion of the PUA domain causes a 4-fold drop in the observed k(cat) (2.8 x 10(-3) s(-1)) and results in a 75-fold increased K(m) for tRNA(Asp)(1.2 x 10(-5) m) compared with full-length TGT. Mutations in tRNA(Asp) altering or abolishing interactions with the PUA domain can compete with wild-type tRNA(Asp) for binding to full-length and truncated TGT enzymes. Whereas the C-terminal domains do not appear to play a role in selection of the modification site, their relevance for enzyme function and their role in vivo remains to be discovered.     

Effects of modification of 4-thiouridine in E. coli tRNA(fMet) on its methyl acceptor activity by thermostable Gm-methylases

tRNA(guanosine-2'-)-methyltransferases (Gm-methylases) isolated from extreme thermophiles, Thermus thermophilus strains HB 27 and HB 8, methylate the 2'-OH of the G18 ribose of the GG sequence in the D loop of tRNA, by recognizing the D "loop-stem" structure as a minimal requirement. To examine the role of the consensus uridine residue at position 8 (U8) adjacent to the D "loop-stem" region in the recognition of Gm-methylase, 4-thiouridine at this position (s4U8) in Escherichia coli tRNAfMet was modified reversibly with S-benzylthioisothiourea (sBTIU) or irreversibly by UV light. The initial velocities of the methylation reaction for the sBTIU-modified and the UV-induced cross-linked tRNAs were decreased to 40 and 30%, respectively, of that of the intact tRNA, but the sBTIU-modified tRNA regained almost full activity on reduction with beta-mercaptoethanol. Although both of the modified tRNAfMetS showed larger Km (although to different extents) and slightly smaller Vmax than the intact tRNAfMet, they retained full activities of methylation with tRNA(adenine-1-)-methyltransferase (m1A-methylase) and of aminoacylation with aminoacyl-tRNA synthetase (ARS) fraction as well, both of which were prepared from T. thermophilus strain HB 27. The 5'-half fragments derived from the sBTIU-modified and cross-linked tRNAfMetS showed methylation efficiency (Vmax/Km) not appreciably different from that of the unmodified 5'-half fragment. These results suggest that the conformation of S4U8 residue of tRNA is deeply involved in the recognition of tRNA by Gm-methylase.     

NMR study of isoleucine transfer RNA from Thermus thermophilus

An NMR and nuclear Overhauser effect (NOE) analysis of Thermus thermophilus tRNAIle1a is presented. This species contains modifications including s2T54 and s4U8 [Horie, N., Hara-Yokoyama, M., Yokoyama, S., Watanabe, K., Kuchino, Y., Nishimura, S., & Miyazawa, T. (1985) Biochemistry 24, 5711-5715]. All the expected secondary and reverse Hoogsteen AU pairs were identified, with one possible exception. The general geometry of the T psi C loop is the same as the Escherichia coli species, and there is NOE evidence for an A9-UA12 triple. Preliminary measurements of solvent exchange rates of internally hydrogen-bonded bases suggest that this tRNA is more stable than previously studied E. coli and yeast tRNAs.     

Role of ribothymidine in the thermal stability of transfer RNA as monitored by proton magnetic resonance.

In order to elucidate the functional role of the modified uridines at position 54 of tRNA, the 270 MHz high-field proton NMR spectra of methionine tRNAs from E. coli, from a mutant thereof, and from T. thermophilus, containing ribothymidine, uridine and 2-thioribothymidine, respectively, have been measured as a function of temperature. A comparison of the NMR melting profiles of the minor nucleosides from these tRNAs shows that the melting temperature of uridine containing tRNA is 6 degrees C lower than that of the wild type tRNA whereas that of the 2-thioribothymidine tRNA is 7 degrees C higher than that of the wild type tRNA. These results, therefore, demonstrate that these modifications serve for stabilization of the tertiary structure of tRNA.     

Sites of methylation of purified transfer ribonucleic acid preparations by enzymes from normal tissues and from tumours induced by dimethylnitrosamine and 1,2-dimethylhydrazine

1. The sites within the tRNA sequence of nucleosides methylated by the action of enzymes from mouse colon, rat kidney and tumours of these tissues acting on tRNA(Asp) from yeast and on tRNA(Glu) (2), tRNA(fMet) and tRNA(Val) (1) from Escherichia coli were determined. 2. The same sites in a particular tRNA were methylated by all of these extracts. Thus tRNA(Glu) (2) was methylated at the cytidine residue at position 48 and the adenosine residue at position 58 from the 5'-end of the molecule; tRNA(Asp) was methylated at the guanosine residue at position 26 from the 5'-end of the molecule; tRNA(fMet) was methylated at the guanosine residues 9 and 27, the cytidine residue 49 and the adenosine residue 59 from the 5'-end; tRNA(Val) (1) was methylated at the guanosine residue 10, the cytidine residue 48 and the adenosine residue 58 from the 5'-end. 3. All of these sites within the clover leaf structure of the tRNA sequence are occupied by a methylated nucleoside in some tRNA species of known sequence. It is concluded that methylation of tRNA from micro-organisms by enzymes from mammalian tissues in vitro probably does accurately represent the specificity of these enzymes in vivo. However, there was no evidence that the tumour extracts, which had considerably greater tRNA methylase activity than the normal tissues, had methylases with altered specificity capable of methylating sites not methylated in the normal tissues.     

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.     

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.     

An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s²U₃₄) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s²U₃₄ nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s²U₃₄ modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.     

Mapping contacts between Escherichia coli alanyl tRNA synthetase and 2' hydroxyls using a complete tRNA molecule

A dual-specific derivative of yeast tRNA(Phe) is described whose features facilitate structure-function studies of tRNAs. This tRNA has been made in three different bimolecular forms that allow modifications to be easily introduced into any position within the molecule. A set of deoxynucleotide substituted versions of this tRNA has been created and used to examine contacts between tRNA and Escherichia coli alanyl-tRNA synthetase, an enzyme previously shown to interact with 2'-hydroxyls in the acceptor stem of the tRNA. Because the present experiments used a full-length tRNA, several contacts were identified that had not been previously found using microhelix substrates. Contacts at similar sites in the T-loop are seen in the cocrystal structure of tRNA(Ser) and Thermus thermophilus seryl-tRNA synthetase.     

An intermediate step in the recognition of tRNA(Asp) by aspartyl-tRNA synthetase

The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNA(Asp) from either T. thermophilus or Escherichia coli reveal a potential intermediate of the recognition process. The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes. While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains. The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 degrees C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state. Based on these data, a pathway for tRNA binding and recognition is proposed.