Elongator tRNA Research Articles

Conditional rather than absolute requirements of the capsid coding sequence for initiation of methionine-independent translation in Plautia stali intestine virus.

The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5' region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5' part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5' part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3' part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes.

Codon-Anticodon Interaction at the P Site Is a Prerequisite for tRNA Interaction with the Small Ribosomal Subunit

The arrival of high resolution crystal structures for the ribosomal subunits opens a new phase of molecular analysis and asks for corresponding analyses of ribosomal function. Here we apply the phosphorothioate technique to dissect tRNA interactions with the ribosome. We demonstrate that a tRNA bound to the P site of non-programmed 70 S ribosomes contacts predominantly the 50 S, as opposed to the 30 S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30 S binding. Protection patterns of tRNAs bound to isolated subunits and programmed 70 S ribosomes were compared. The results suggest the presence of a movable domain in the large ribosomal subunit that carries tRNA and reveal that only approximately 15% of a tRNA, namely residues 30 +/- 1 to 43 +/- 1, contact the 30 S subunit of programmed 70 S ribosomes, whereas the remaining 85% make contact with the 50 S subunit. Identical protection patterns of two distinct elongator tRNAs at the P site were identified as tRNA species-independent phosphate backbone contacts. The sites of protection correlate nicely with the predicted ribosomal-tRNA contacts deduced from a 5.5-A crystal structure of a programmed 70 S ribosome, thus refining which ribosomal components are critical for tRNA fixation at the P site.

The large subunit of initiation factor aIF2 is a close structural homologue of elongation factors.

The heterotrimeric factor e/aIF2 plays a central role in eukaryotic/archaeal initiation of translation. By delivering the initiator methionyl-tRNA to the ribosome, e/aIF2 ensures specificity of initiation codon selection. The three subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus abyssi could be overproduced in Escherichia coli. The beta and gamma subunits each contain a tightly bound zinc. The large gamma subunit is shown to form the structural core for trimer assembly. The crystal structures of aIF2gamma, free or complexed to GDP-Mg(2+) or GDPNP-Mg(2+), were resolved at resolutions better than 2 A. aIF2gamma displays marked similarities to elongation factors. A distinctive feature of e/aIF2gamma is a subdomain containing a zinc-binding knuckle. Examination of the nucleotide-complexed aIF2gamma structures suggests mechanisms of action and tRNA binding properties similar to those of an elongation factor. Implications for the mechanism of translation initiation in both eukarya and archaea are discussed. In particular, positioning of the initiator tRNA in the ribosomal A site during the search for the initiation codon is envisaged.

Unleashing yeast genetics on a factor-independent mechanism of internal translation initiation.

Translational control of gene expression is generally exerted in the initiation phase of protein synthesis. The reactions of the initiation pathway bring the ribosome to the mRNA and place the initiation codon in the peptidyl (P) site of the ribosome, base-paired with the anticodon of methionyl initiator tRNA (Met-tRNA). The aminoacyl (A) site in the ribosome is left open to accept the elongator tRNA that decodes the second codon in the ORF. Formation of this 80S initiation complex in eukaryotic cells depends on 11 or more soluble initiation factors (eIFs), identified primarily through biochemical studies of translation in rabbit reticulocyte extracts. Most of these eIFs are essential for normal cell growth in budding yeast, confirming their importance in vivo (1). Nevertheless, a number of mRNAs contain specialized regulatory elements that allow them to dispense with one or more eIFs during translation initiation. These elements, called internal ribosome entry sites (IRESs), recruit the ribosome directly to the initiation region of the mRNA. Their independence of certain eIFs allows viral mRNAs containing IRESs to be translated in infected cells where one or more eIFs have been impaired, either by the virus to provide a selective advantage for its mRNAs or by the host to impede virus gene expression. The most striking instance of this strategy is provided by cricket paralysis virus (CrPV) mRNA, which seems to be translated without any eIFs or even Met-tRNA (2). The work reported by Sarnow and colleagues (3) in this issue of PNAS shows that the unique CrPV initiation mechanism can operate in budding yeast cells, so long as the canonical initiation pathway involving initiation factor 2 (eIF2) has been impaired. These findings open up the possibility of using powerful yeast genetics to dissect the details of this remarkable mechanism and to define the factors that modulate its efficiency in living cells.

Why Termination tRNAs Have Not Been Found? Because They Are Hidden in the Large Ribosomal RNA (A Hypothesis)

It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such recognition to make it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.

Junction phosphate is derived from the precursor in the tRNA spliced by the archaeon Haloferax volcanii cell extract.

RNA splicing in archaea requires at least an endonuclease and a ligase, as is the case for the splicing of eukaryal nuclear tRNAs. Splicing endonucleases from archaea and eukarya are homologous, although they differ in subunit composition and substrate recognition properties. However, they all produce 2',3' cyclic phosphate and 5'-hydroxyl termini. An in vitro-transcribed, partial intron-deleted Haloferax volcanii elongator tRNA(Met) has been used to study splicing by H. volcanii cell extracts. Substrates and products were analyzed by nearest neighbor analyses using nuclease P1 and RNase T2, and fingerprinting analyses using acid-urea gels in the first dimension and gradient thin layer chromatography in the second dimension. The results suggest that 2',3' cyclic phosphate at the 3' end of the 5' exon is converted into the splice junction phosphate forming a 3',5'-phosphodiester linkage. This resembles the animal cell type systems where the junction phosphate preexists in the transcript, and differs from yeast type systems, where GTP is the source of junction phosphate.

Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae.

The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.

Function of the extra 5'-phosphate carried by histidine tRNA.

Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1. This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase. Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase. In the case of the reaction of E. coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable. Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His). Recognition of the +1 phosphate of tRNA(His) by peptidyl-tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs.

Splicing of arabidopsis tRNA(Met) precursors in tobacco cell and wheat germ extracts.

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA(GpsiA(Tyr)) and elongator tRNA(CmAU(Met)) contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA(Tyr). Here we have studied the expression of an Arabidopsis elongator tRNA(Met) gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA(Met) precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA(Met) to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3' and 5' splice sites and of a structured intron for pre-tRNA(Met) splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA(Met) splicing and that a highly structured intron is indispensable for pre-tRNA(Met) splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA(Met) gene, is efficiently processed and spliced in both plant extracts.

Recognition of the initiator tRNA by the Pseudomonas aeruginosa methionyl-tRNA formyltransferase: importance of the base-base mismatch at the end of the acceptor stem

Formylation of the initiator methionyl-tRNA (Met-tRNA fMet) in eubacteria is catalyzed by methionyl-tRNA formyltransferase (MTF). Features of the Escherichia coli tRNA fMet that are important for formylation are the base-base mismatch between nucleotides 1 and 72, and the second and third base pairs of the acceptor stem. The base-base mismatch is the most crucial formylation determinant in the E. coli tRNA fMet. However, it is not known whether this feature is also important for formylation of other eubacterial tRNA fMet. We cloned the Pseudomonas aeruginosa MTF gene by complementation of an E. coli MTF mutant strain with a genomic library, and investigated the catalytic properties and substrate specificity of the enzyme. The results show that the P. aeruginosa and E. coli enzymes have comparable affinities for the tRNA fMet and N 10-formyltetrahydrofolate (fTHF) substrates. Overproduction of the P. aeruginosa MTF rescued the initiator activity of an E. coli formylation-defective tRNA fMet with a base pair between nucleotides 1 and 72, indicating that the base-base mismatch is utilized by the P. aeruginosa MTF for recognition of the tRNA fMet. Therefore, this feature may be used by MTFs from other eubacteria to distinguish the initiator from elongator tRNAs.

Recognition of the initiator tRNA by the Pseudomonas aeruginosa methionyl-tRNA formyltransferase: importance of the base-base mismatch at the end of the acceptor stem.

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) in eubacteria is catalyzed by methionyl-tRNA formyltransferase (MTF). Features of the Escherichia coli tRNAfMet that are important for formylation are the base-base mismatch between nucleotides 1 and 72, and the second and third base pairs of the acceptor stem. The base-base mismatch is the most crucial formylation determinant in the E. coli tRNAfMet. However, it is not known whether this feature is also important for formylation of other eubacterial tRNAfMet. We cloned the Pseudomonas aeruginosa MTF gene by complementation of an E. coli MTF mutant strain with a genomic library, and investigated the catalytic properties and substrate specificity of the enzyme. The results show that the P. aeruginosa and E. coli enzymes have comparable affinities for the tRNAfMet and N10-formyltetrahydrofolate (fTHF) substrates. Overproduction of the P. aeruginosa MTF rescued the initiator activity of an E. coli formylation-defective tRNAfMet with a base pair between nucleotides 1 and 72, indicating that the base-base mismatch is utilized by the P. aeruginosa MTF for recognition of the tRNAfMet. Therefore, this feature may be used by MTFs from other eubacteria to distinguish the initiator from elongator tRNAs.

Selenocysteine inserting tRNAs: an overview

One of the recent discoveries in protein biosynthesis was the finding that selenocysteine, the 21st amino acid, is cotranslationally inserted into polypeptides under the direction of a UGA codon assisted by a specific structural signal in the mRNA. The key to selenocysteine biosynthesis and insertion is a special tRNA species, tRNA(Sec). The formation of selenocysteine from serine represents an interesting tRNA-mediated amino acid transformation. tRNA(Sec) (or the gene encoding it) has been found over all three domains of life. It displays a number of unique features that designate it a selenocysteine-inserting tRNA and differentiate it from canonical elongator tRNAs. Although there are still some uncertainties concerning the precise secondary and tertiary structures of eukaryal tRNA(Sec), the major identity determinant for selenocysteine biosynthesis and insertion appears to be the 13 bp long extended acceptor arm. In addition the core of the 3D structure of these tRNAs is different from that of class II tRNAs like tRNA(Sec). The biological implications of these structural differences still remain to be fully understood.

Receptor site for the 5'-phosphate of elongator tRNAs governs substrate selection by peptidyl-tRNA hydrolase.

Eubacterial peptidyl-tRNA hydrolase (PTH) recycles all N-blocked aminoacyl-tRNA molecules but initiator formyl-methionyl-tRNAfMet, the acceptor helix of which is characterized by a 1-72 mismatch. Positive selection by PTH of noninitiator tRNA molecules with a full 1-72 base pair is abolished, however, upon the removal of the 5'-phosphate. The tRNA 5'-phosphate plays therefore the role of a relay between the enzyme and the status of the 1-72 base pair. In this study, the receptor site for the 5'-phosphate of elongator peptidyl-tRNAs and the position at the surface of PTH of the 3'-end of complexed peptidyl-tRNA are identified by site-directed mutagenesis experiments. The former site comprehends two cationic side chains (K105 and R133) which are likely to clamp the phosphate. The second corresponds to a four asparagine cluster (N10, N21, N68, and N114). By using these two positional constraints, the acceptor arm of elongation factor Tu-bound Phe-tRNAPhe could be docked to PTH. Contacts involve the acceptor and TPsiC stems. By comparing the obtained 3D model to that of EF-Tu:Phe-tRNAPhe crystalline complex in which the 5'-phosphate of the ligand also lies between a K and an R side chain, we propose that, in both systems, the capacity of the 5'-phosphate of a tRNA to reach or not a receptor site is the main identity element governing generic selection of elongator tRNAs. On the other hand, while the 1-72 mismatch acts as an antideterminant for PTH or EF-Tu recognition, it behaves as a positive determinant for the formylation of initiator Met-tRNAfMet.

Crystal structure of methionyl-tRNAfMet transformylase complexed with the initiator formyl-methionyl-tRNAfMet.

The crystal structure of Escherichia coli methionyl-tRNAfMet transformylase complexed with formyl-methionyl-tRNAfMet was solved at 2.8 A resolution. The formylation reaction catalyzed by this enzyme irreversibly commits methionyl-tRNAfMet to initiation of translation in eubacteria. In the three-dimensional model, the methionyl-tRNAfMet formyltransferase fills in the inside of the L-shaped tRNA molecule on the D-stem side. The anticodon stem and loop are away from the protein. An enzyme loop is wedged in the major groove of the acceptor helix. As a result, the C1-A72 mismatch characteristic of the initiator tRNA is split and the 3' arm bends inside the active centre. This recognition mechanism is markedly distinct from that of elongation factor Tu, which binds the acceptor arm of aminoacylated elongator tRNAs on the T-stem side.

Activation of microhelix charging by localized helix destabilization.

We report that aminoacylation of minimal RNA helical substrates is enhanced by mismatched or unpaired nucleotides at the first position in the helix. Previously, we demonstrated that the class I methionyl-tRNA synthetase aminoacylates RNA microhelices based on the acceptor stem of initiator and elongator tRNAs with greatly reduced efficiency relative to full-length tRNA substrates. The cocrystal structure of the class I glutaminyl-tRNA synthetase with tRNAGln revealed an uncoupling of the first (1.72) base pair of tRNAGln, and tRNAMet was proposed by others to have a similar base-pair uncoupling when bound to methionyl-tRNA synthetase. Because the anticodon is important for efficient charging of methionine tRNA, we thought that 1.72 distortion is probably effected by the synthetase-anticodon interaction. Small RNA substrates (minihelices, microhelices, and duplexes) are devoid of the anticodon triplet and may, therefore, be inefficiently aminoacylated because of a lack of anticodon-triggered acceptor stem distortion. To test this hypothesis, we constructed microhelices that vary in their ability to form a 1.72 base pair. The results of kinetic assays show that microhelix aminoacylation is activated by destabilization of this terminal base pair. The largest effect is seen when one of the two nucleotides of the pair is completely deleted. Activation of aminoacylation is also seen with the analogous deletion in a minihelix substrate for the closely related isoleucine enzyme. Thus, for at least the methionine and isoleucine systems, a built-in helix destabilization compensates in part for the lack of presumptive anticodon-induced acceptor stem distortion.

On the conformation of the anticodon loops of initiator and elongator methionine tRNAs

The solution conformations of analogues of initiator and elongator tRNA anticodon stem-loops have been compared by NMR. The data indicate that both have conformations closely similar to those reported for crystalline elongator tRNAs. The two loops differ in their dynamics, however: that of the elongator analogue is more flexible than its initiator counterpart. The anticodon stem-loops of initiator tRNAs are more likely to be distinguished from those of elongator tRNAs during initiation on the basis of their distinctive stem sequences, than they are by differences in the conformations of their anticodon loops.

Analysis of interactions between the Codon-Anticodon duplexes within the ribosome: their role in translation

Computer graphics simulation of interactions between the codon-anticodon duplexes formed by normal elongator tRNAs at the ribosomal A, P and E-sites (the AP and PE interduplex interactions) was made. This demonstrated that only the correct duplexes at the A-site are compatible with the AP interduplex interaction. The selection of synonymous codons and anticodon wobble bases, together with the AP interduplex interaction, prevents frameshifting. In the absence of this interaction the efficiency of the selection falls off sharply. This suggests that the AP interduplex interaction should be retained during translocation and in the post-translocation state, i.e. the PE interduplex interaction that is identical with that of AP should exist to avoid frameshifting. In such a model the P-site duplex provides an indirect linkage between the A and E-site duplexes. The indirect linkage prohibits the simultaneous existence of the A and E-site duplexes. The wobble pairs of the P and E-site duplexes can affect the rate of the A-site occupation via the AP interduplex interaction and the AE interduplex indirect linkage. It is demonstrated that frameshifting can occur from the AP or PE codon-anticodon complex destabilization caused, for example, by small mobility of the wobble pairs, misreading of the codon, unmodified adenine and guanine at tRNA positions 34 (wobble) and 37, respectively. The results obtained can be subjected to direct experimental tests.

Plant nuclear tRNA(Met) genes are ubiquitously interrupted by introns.

We have isolated three independent clones for nuclear elongator tRNA(Met) genes from an Arabidopsis DNA library using a tRNA(Met)-specific probe generated by PCR. Each of the coding sequences for tRNAMet in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA(Met) is dispersed at at least eight loci in the genome. The unspliced precursor tRNA(Met) intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA(Met) genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5'-distal end of each intron.

Mutations at Two Invariant Nucleotides in the 3′-Minor Domain of Escherichia coli 16 S rRNA Affecting Translational Initiation and Initiation Factor 3 Function

We have investigated the highly conserved GAUCA sequence of small subunit ribosomal RNA. Within this region, the invariant nucleotides G1530 and A1531 of Escherichia coli 16 S rRNA were mutagenized to A1530/G1531. These base changes caused a lethal phenotype when expressed from a high copy number plasmid. In low copy number plasmids, the mutant ribosomes had limited effects when expressed in vivo but caused significant deficiencies in translation in vitro, affecting enzymatic tRNA binding, non-enzymatic tRNA binding, subunit association, and initiation factor 3 (IF3) binding. Mutant 30 S ribosomal subunits showed a 10-fold decrease in affinity for IF3 as compared to wild-type subunits but showed an increased affinity for IF3 when in 70 S ribosomes. Additionally, IF3 did not promote dissociation of 70 S ribosomes, which had mutated subunits as monitored by light-scattering experiments. However, extension inhibition experiments (toeprinting) showed that IF3 retained its ability to discriminate between initiator and elongator tRNAs on mutated subunits. The results indicate that the two functions of IF3, tRNA discrimination and subunit dissociation, are separable and that the invariant nucleotides are important for correct subunit function during initiation.

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.