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Biogenic polyamines are natural aliphatic polycations formed from amino acids by biochemical pathways that are highly conserved from bacteria to humans. Their cellular concentrations are carefully regulated and dysregulation causes severe cell growth defects. Polyamines have high affinity for nucleic acids and are known to interact with mRNA, tRNA and rRNA to stimulate the translational machinery, but the exact molecular mechanism(s) for this stimulus is still unknown. Here we exploit that Escherichia coli is viable in the absence of polyamines, including the universally conserved putrescine and spermidine. Using global macromolecule labelling approaches we find that ribosome efficiency is reduced by 50-70% in the absence of polyamines and this reduction is caused by slow translation elongation speed. The low efficiency causes rRNA and multiple tRNA species to be overproduced in the absence of polyamines, suggesting an impact on the feedback regulation of stable RNA transcription. Importantly, we find that polyamine deficiency affects both tRNA levels and tRNA modification patterns. Specifically, a large fraction of tRNA his , tRNA tyr and tRNA asn lack the queuosine modification in the anticodon "wobble" base, which can be reversed by addition of polyamines to the growth medium. In conclusion, we demonstrate that polyamines are needed for modification of specific tRNA, possibly by facilitating the interaction with modification enzymes., Competing Interests: Conflict of interest statement None declared., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tuberculosis (TB) is a severe disease caused by Mycobacterium tuberculosis (M. tb) and the well-characterized M. tb MazE/F proteins play important roles in stress adaptation. Recently, the MazF-mt9 toxin has been found to display endonuclease activities towards tRNAs but the mechanism is unknown. We hereby present the crystal structure of apo-MazF-mt9. The enzyme recognizes tRNA Lys with a central UUU motif within the anticodon loop, but is insensitive to the sequence context outside of the loop. Based on our crystallographic and biochemical studies, we identified key residues for catalysis and proposed the potential tRNA-binding site., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA(Gln) for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA(Gln) and tRNA(Glu) with glutamate. This ancient GluRS also separately differentiated to exclude tRNA(Gln) as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA(Gln) and tRNA(Glu) recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The 2-thiouridine (s(2)U) at the wobble position of certain bacterial and eukaryotic tRNAs enhances aminoacylation kinetics, assists proper codon-anticodon base pairing at the ribosome A-site, and prevents frameshifting during translation. By mass spectrometry of affinity-purified native Escherichia coli tRNA1(Gln)UUG, we show that the complete modification at the wobble position 34 is 5-carboxyaminomethyl-2-thiouridine (cmnm(5)s(2)U). The crystal structure of E. coli glutaminyl-tRNA synthetase (GlnRS) bound to native tRNA1(Gln) and ATP demonstrates that cmnm(5)s(2)U34 improves the order of a previously unobserved 11-amino-acid surface loop in the distal β-barrel domain of the enzyme and imparts other local rearrangements of nearby amino acids that create a binding pocket for the 2-thio moiety. Together with previously solved structures, these observations explain the degenerate recognition of C34 and modified U34 by GlnRS. Comparative pre-steady-state aminoacylation kinetics of native tRNA1(Gln), synthetic tRNA1(Gln) containing s(2)U34 as sole modification, and unmodified wild-type and mutant tRNA1(Gln) and tRNA2(Gln) transcripts demonstrates that the exocyclic sulfur moiety improves tRNA binding affinity to GlnRS 10-fold compared with the unmodified transcript and that an additional fourfold improvement arises from the presence of the cmnm(5) moiety. Measurements of Gln-tRNA(Gln) interactions at the ribosome A-site show that the s(2)U modification enhances binding affinity to the glutamine codons CAA and CAG and increases the rate of GTP hydrolysis by E. coli EF-Tu by fivefold., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human tRNA(Lys3)(UUU) (htRNA(Lys3)(UUU)) decodes the lysine codons AAA and AAG during translation and also plays a crucial role as the primer for HIV-1 (human immunodeficiency virus type 1) reverse transcription. The posttranscriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)), 2-methylthio-N(6)-threonylcarbamoyladenosine (ms(2)t(6)A(37)), and pseudouridine (Ψ(39)) in the tRNA's anticodon domain are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, we determined the structure and function of this tRNA's anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39)). Ribosome binding assays in vitro revealed that the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound AAA and AAG codons, whereas binding of the unmodified ASL(Lys3)(UUU) was barely detectable. The UV hyperchromicity, the circular dichroism, and the structural analyses indicated that Ψ(39) enhanced the thermodynamic stability of the ASL through base stacking while ms(2)t(6)A(37) restrained the anticodon to adopt an open loop conformation that is required for ribosomal binding. The NMR-restrained molecular-dynamics-derived solution structure revealed that the modifications provided an open, ordered loop for codon binding. The crystal structures of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm(5)s(2)U(34) and ms(2)t(6)A(37) participate in the stability of the anticodon-codon interaction. Importantly, the mcm(5)s(2)U(34)·G(3) wobble base pair is in the Watson-Crick geometry, requiring unusual hydrogen bonding to G in which mcm(5)s(2)U(34) must shift from the keto to the enol form. The results unambiguously demonstrate that modifications pre-structure the anticodon as a key prerequisite for efficient and accurate recognition of cognate and wobble codons., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Three of six arginine codons are read by two tRNA(Arg) isoacceptors in Escherichia coli. The anticodon stem and loop of these isoacceptors (ASL(Arg1,2)) differs only in that the position 32 cytidine of tRNA(Arg1) is posttranscriptionally modified to 2-thiocytidine (s(2)C(32)). The tRNA(Arg1,2) are also modified at positions 34 (inosine, I(34)) and 37 (2-methyladenosine, m(2)A(37)). To investigate the roles of modifications in the structure and function, we analyzed six ASL(Arg1,2) constructs differing in their array of modifications by spectroscopy and codon binding assays. Thermal denaturation and circular dichroism spectroscopy indicated that modifications contribute thermodynamic and base stacking properties, resulting in more order but less stability. NMR-derived structures of the ASL(Arg1,2) showed that the solution structures of the ASLs were nearly identical. Surprisingly, none possessed the U-turn conformation required for effective codon binding on the ribosome. Yet, all ASL(Arg1,2) constructs efficiently bound the cognate CGU codon. Three ASLs with I(34) were able to decode CGC, whereas only the singly modified ASL(Arg1,2)(ICG) with I(34) was able to decode CGA. The dissociation constants for all codon bindings were physiologically relevant (0.4-1.4 μM). However, with the introduction of s(2)C(32) or m(2)A(37) to ASL(Arg1,2)(ICG), the maximum amount of ASL bound to CGU and CGC was significantly reduced. These results suggest that, by allowing loop flexibility, the modifications modulate the conformation of the ASL(Arg1,2), which takes one structure free in solution and two others when bound to the cognate arginyl-tRNA synthetase or to codons on the ribosome where modifications reduce or restrict binding to specific codons., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA(Lys3)(UUU) as the primer for reverse transcription. NCp7 also remodels the htRNA's amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA's unique composition of post-transcriptional modifications on NCp7 recognition of htRNA(Lys3)(UUU), the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL(Lys3)) were studied. NCp7 bound the hASL(Lys3)(UUU) modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm(5)s(2)U(34)) and 2-methylthio-N(6)-threonylcarbamoyladenosine at position-37 (ms(2)t(6)A(37)) with a considerably higher affinity than the unmodified hASL(Lys3)(UUU) (K(d)=0.28±0.03 and 2.30±0.62 μM, respectively). NCp7 denatured the structure of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) more effectively than that of the unmodified hASL(Lys3)(UUU). Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K(d)=0.55±0.10 μM) and specificity for the ASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37) comparable to that of NCp7. The peptides recognized a t(6)A(37)-modified ASL with an affinity (K(d)=0.60±0.09 μM) comparable to that for hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37), indicating a preference for the t(6)A(37) modification. Significantly, one of the peptides was capable of relaxing the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA(Lys3)(UUU) have been found to be important determinants of NCp7's recognition prior to the tRNA(Lys3)(UUU) being annealed to the viral genome as the primer of reverse transcription., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNA(Met)(CAU), to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl- (A-) sites of the ribosome. The hmtRNA(Met)(CAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f(5)C(34)), and a cytidine substituting for the invariant uridine at position 33 of the canonical U-turn in tRNAs. The structure of the tRNA anticodon stem and loop domain (hmtASL(Met)(CAU)), determined by NMR restrained molecular modeling, revealed how the f(5)C(34) modification facilitates the decoding of AUA at the P- and the A-sites. The f(5)C(34) defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASL(Met)(CAU). The hmtASL(Met)(CAU) exhibited a C-turn conformation that has some characteristics of the U-turn motif. Codon binding studies with both Escherichia coli and bovine mitochondrial ribosomes revealed that the f(5)C(34) facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL binding kinetics. Mitochondrial translation by many organisms, including humans, sometimes initiates with the universal isoleucine codons AUU and AUC. The f(5)C(34) enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f(5)C(34), expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Pathogenic mutations in mitochondrial tRNAs are 6.5 times more frequent than in other mitochondrial genes. This suggests that tRNA mutations perturb more than one function. A potential additional tRNA gene function is that of templating for antisense tRNAs. Pathogenic mutations weaken cloverleaf secondary structures of sense tRNAs. Analyses here show similar effects for most antisense tRNAs, especially after adjusting for associations between sense and antisense cloverleaf stabilities. These results imply translational activity by antisense tRNAs. For sense tRNAs Ala and Ser UCN, pathogenicity associates as much with sense as with antisense cloverleaf formation. For tRNA Pro, pathogenicity seems associated only with antisense, not sense tRNA cloverleaf formation. Translational activity by antisense tRNAs is expected for the 11 antisense tRNAs processed by regular sense RNA maturation, those recognized by their cognate amino acid's tRNA synthetase, and those forming relatively stable cloverleaves as compared to their sense counterpart. Most antisense tRNAs probably function routinely in translation and extend the tRNA pool (extension hypothesis); others do not (avoidance hypothesis). The greater the expected translational activity of an antisense tRNA, the more pathogenic mutations weaken its cloverleaf secondary structure. Some evidence for RNA interference, a more classical role for antisense tRNAs, exists only for tRNA Ser UCN. Mutation pathogenicity probably frequently results from a mixture of effects due to sense and antisense tRNA translational activity for many mitochondrial tRNAs. Genomic studies should routinely explore for translational activity by antisense tRNAs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K(m)=47.1muM and a k(cat)=0.151s(-1), which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA(Cys) with the GCA anticodon (26.9muM and 0.111s(-1)). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNAs. Seryl-tRNA synthetase is a class II synthetase, which depends on rather few and simple identity elements in tRNA(Ser) to determine the amino acid specificity. tRNA(Ser) acceptor stem microhelices can be aminoacylated with serine, which makes this part of the tRNA a valuable tool for investigating the structural motifs in a tRNA(Ser)-seryl-tRNA synthetase complex. A 1.8A-resolution tRNA(Ser) acceptor stem crystal structure was superimposed to a 2.9A-resolution crystal structure of a tRNA(Ser)-seryl-tRNA synthetase complex for a visualization of the binding environment of the tRNA(Ser) microhelix.

The genetic code is degenerate, in that 20 amino acids are encoded by 61 triplet codes. In 1966, Francis Crick hypothesized that the cell's limited number of tRNAs decoded the genome by recognizing more than one codon. The ambiguity of that recognition resided in the third base-pair, giving rise to the Wobble Hypothesis. Post-transcriptional modifications at tRNA's wobble position 34, especially modifications of uridine 34, enable wobble to occur. The Modified Wobble Hypothesis proposed in 1991 that specific modifications of a tRNA wobble nucleoside shape the anticodon architecture in such a manner that interactions were restricted to the complementary base plus a single wobble pairing for amino acids with twofold degenerate codons. However, chemically different modifications at position 34 would expand the ability of a tRNA to read three or even four of the fourfold degenerate codons. One foundation of Crick's Wobble Hypothesis was that a near-constant geometry of canonical base-pairing be maintained in forming all three base-pairs between the tRNA anticodon and mRNA codon on the ribosome. In accepting an aminoacyl-tRNA, the ribosome requires maintenance of a specific geometry for the anticodon-codon base-pairing. However, it is the post-transcriptional modifications at tRNA wobble position 34 and purine 37, 3'-adjacent to the anticodon, that pre-structure the anticodon domain to ensure the correct codon binding. The modifications create both the architecture and the stability needed for decoding through restraints on anticodon stereochemistry and conformational space, and through selective hydrogen bonding. A physicochemical understanding of modified nucleoside contributions to the tRNA anticodon domain architecture and its decoding of the genome has advanced RNA world evolutionary theory, the principles of RNA chemistry, and the application of this knowledge to the introduction of new amino acids to proteins.

During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNA(GAG)(Leu). As predicted, the charged level of tRNA(GAG)(Leu) increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNA(UAG)(Leu) increased unexpectedly. To remove this apparent inconsistency between theory and experiment we postulated a previously unknown common codon for tRNA(GAG)(Leu) and tRNA(UAG)(Leu). Subsequently, we demonstrated that the tRNA(GAG)(Leu) codon CUU is, in fact, read also by tRNA(UAG)(Leu), due to a uridine-5-oxyacetic acid modification.

Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce S-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.

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.

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix., ((c) 2002 Elsevier Science Ltd.)

Accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetase is essential for the fidelity of protein synthesis. For Methanococcus jannaschii tRNA(Pro), accuracy is difficult because the cognate prolyl-tRNA synthetase also recognizes and aminoacylates tRNA(Cys) with cysteine. We show here that the unmodified transcript of M. jannaschii tRNA(Pro) is indeed mis-acylated with cysteine. However, the origin of mis-charging is not at the anticodon or acceptor stem, the two hotspots for tRNA(Pro) and tRNA(Cys) identity determinants. Instead, replacement of the D loop in the tRNA core with that of tRNA(Cys) suppresses mis-charging with cysteine without compromising the activity of aminoacylation with proline. The reduced level of cysteine activity of the chimera is not due an editing response of the synthetase and is consistent with a relaxed sensitivity of the tRNA to the analog thiaproline in aminoacylation with cysteine. We suggest that mis-acylation is not due to the presence of cysteine determinants, but to a mis-placed 3' end into the cysteine catalytic site that activates and transfers cysteine to the tRNA. Prevention of mis-placement by alteration of the core structure or by nucleotide modifications in the tRNA illustrates a novel strategy of the dual-specificity synthetase., (Copyright 2002 Elsevier Science Limited.)

Naturally occurring tRNA mutants are known that suppress +1 frameshift mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis. In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general strategy to select tRNAs with the ability to suppress four-base codons from a library of tRNAs with randomized 8 or 9 nt anticodon loops. Our selectants included both known and novel suppressible four-base codons and resulted in a set of very efficient, non-cross-reactive tRNA/four-base codon pairs for AGGA, UAGA, CCCU and CUAG. The most efficient four-base codon suppressors had Watson-Crick complementary anticodons, and the sequences of the anticodon loops outside of the anticodons varied with the anticodon. Additionally, four-base codon reporter libraries were used to identify "shifty" sites at which +1 frameshifting is most favorable in the absence of suppressor tRNAs in Escherichia coli. We intend to use these tRNAs to explore the limits of unnatural polypeptide biosynthesis, both in vitro and eventually in vivo. In addition, this selection strategy is being extended to identify novel five- and six-base codon suppressors., (Copyright 2001 Academic Press.)

Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer. Recombinant tRNA(Lys)3 was previously overexpressed in Escherichia coli, 15N-labelled and purified for NMR studies. It was shown to be functional for priming of HIV-1 reverse transcription. Using heteronuclear 2D and 3D NMR, we have been able to assign almost all the imino groups in the helical regions and involved in the tertiary base interactions of tRNA(Lys)3. This crucial step enabled us to address the question of the annealing mechanism of tRNA(Lys)3 by the nucleocapsid protein (NC) using heteronuclear NMR. Moreover, structural aspects of the tRNA(Lys)3/(12-53)NCp7 interaction have been characterised. The (12-53)NCp7 protein binds preferentially to the inside of the L-shape of the tRNA(Lys)3, on the acceptor and D stems, and at the level of the tertiary interactions between the D and T-psi-C loops. (12-53)NCp7 binding does not induce the melting of any single base-pair or unwinding of the tRNA(Lys)3 helical domains. Moreover, NMR provides a unique means to investigate the base-pairs that are destabilised by (12-53)NCp7 binding. Indeed, the measurements of the longitudinal relaxation time T1 and of the exchange time of the imino protons revealed two major regions sensitive to catalysis by the protein, namely the G6-U67 and T54(A58) pairs. It is interesting that for the biological role of the NC protein, these pairs could be the starting points of the tRNA melting required for the hybridisation to the viral RNA.

Functional and structural similarities between tRNA and eukaryotic class 1 release factors (eRF1) described previously, provide evidence for the molecular mimicry concept. This concept is supported here by the demonstration of a genetic interaction between eRF1 and the decoding region of the ribosomal RNA, the site of tRNA-mRNA interaction. We show that the conditional lethality caused by a mutation in domain 1 of yeast eRF1 (P86A), that mimics the tRNA anticodon stem-loop, is rescued by compensatory mutations A1491G (rdn15) and U1495C (hyg1) in helix 44 of the decoding region and by U912C (rdn4) and G886A (rdn8) mutations in helix 27 of the 18 S rRNA. The rdn15 mutation creates a C1409-G1491 base-pair in yeast rRNA that is analogous to that in prokaryotic rRNA known to be important for high-affinity paromomycin binding to the ribosome. Indeed, rdn15 makes yeast cells extremely sensitive to paromomycin, indicating that the natural high resistance of the yeast ribosome to paromomycin is, in large part, due to the absence of the 1409-1491 base-pair. The rdn15 and hyg1 mutations also partially compensate for inactivation of the eukaryotic release factor 3 (eRF3) resulting from the formation of the [PSI+] prion, a self-reproducible termination-deficient conformation of eRF3. However, rdn15, but not hyg1, rescues the conditional cell lethality caused by a GTPase domain mutation (R419G) in eRF3. Other antisuppressor rRNA mutations, rdn2(G517A), rdn1T(C1054T) and rdn12A(C526A), strongly inhibit [PSI+]-mediated stop codon read-through but do not cure cells of the [PSI+] prion. Interestingly, cells bearing hyg1 seem to enable [PSI+] strains to accumulate larger Sup35p aggregates upon Sup35p overproduction, suggesting a lower toxicity of overproduced Sup35p when the termination defect, caused by [PSI+], is partly relieved., (Copyright 2001 Academic Press.)

The U-turn is a well-known RNA motif characterized by a sharp reversal of the RNA backbone following a single-stranded uridine base. In experimentally determined U-turn motifs, the nucleotides 3' to the turn are frequently involved in tertiary interactions, rendering this motif particularly attractive in RNA modeling and functional studies. The U-turn signature is composed of an UNR sequence pattern flanked by a Y:Y, Y:A (Y=pyrimidine) or G:A base juxtaposition. We have identified 33 potential UNR-type U-turns and 25 related GNRA-type U-turns in a large set of aligned 16 S and 23 S rRNA sequences. U-turn candidates occur in hairpin loops (34 times) as well as in internal and multi-stem loops (24 times). These are classified into ten families based on loop type, sequence pattern (UNR or GNRA) and the nature of the closing base juxtaposition. In 13 cases, the bases on the 3' side of the turn, or on the immediate 5' side, are involved in tertiary covariations, making these sites strong candidates for tertiary interactions., (Copyright 2000 Academic Press.)

The crystal structure of aspartyl-tRNA synthetase from Escherichia coli has been determined to a resolution of 2.7 A. The structure is compared to the same enzyme co-crystallized with tRNA(Asp) and containing aspartyl adenylate or ATP. The asymmetric unit contains three monomers of the enzyme. While most parts of the protein show no significant differences in the three monomers, a few regions cannot be superimposed. Those regions are characterized by a high B-factor, and consist mostly of loops that make contacts with the tRNA in the complexes. The flexibility of the protein is seen at a global level, by the observation of a 10 to 15 degrees rotation of the N-terminal and insertion domains upon tRNA binding, and at the level of the individual amino acid residues, by main-chain and side-chain rearrangements. In contrast to these induced-fit conformational changes, a few residues essential for the tRNA anticodon or aspartyl-adenylate recognition exist in a predefined conformation, ensured by specific interactions within the protein., (Copyright 2000 Academic Press.)

Aminoacyl-tRNA synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA(Asp). The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module., (Copyright 2000 Academic Press.)

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., (Copyright 2000 Academic Press.)

One of the requirements for engineering expansion of the genetic code is a unique codon which is available for specifying the new amino acid. The potential of the quadruplet UAGA in Escherichia coli to specify a single amino acid residue in the presence of a mutant tRNA(Leu) molecule containing the extra nucleotide, U, at position 33.5 of its anticodon loop has been examined. With this mRNA-tRNA combination and at least partial inactivation of release factor 1, the UAGA quadruplet specifies a leucine residue with an efficiency of 13 to 26 %. The decoding properties of tRNA(Leu) with U at position 33.5 of its eight-membered anticodon loop, and a counterpart with A at position 33.5, strongly suggest that in both cases their anticodon loop bases stack in alternative conformations. The identity of the codon immediately 5' of the UAGA quadruplet influences the efficiency of quadruplet translation via the properties of its cognate tRNA. When there is the potential for the anticodon of this tRNA to dissociate from pairing with its codon and to re-pair to mRNA at a nearby 3' closely matched codon, the efficiency of quadruplet translation at UAGA is reduced. Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet., (Copyright 2000 Academic Press.)

New insights on the origin of the genetic code, based on the analysis of the physico-chemical properties of its molecular constituents (RNA and amino acids), are reported in this paper. We point out a symmetry in the genetic code table and show that it can be explained by the nature of the anticodon-codon interaction. The importance of the strength of this interaction is examined and a correlation is found between the free-energy change (DeltaG(0)) of anticodon-codon association and the volume of the corresponding amino acids. This correlation is investigated in conjunction with the well-known one linking the hydrophobicity of the anticodons with that of the amino acids. We show that they can be considerated separately and that the energy vs. volume correlation may be explained by the process implicating the peptide bond formation between two successive amino acids during translation. This interpretation is supported by a statistical pattern of bases (purines or pyrimidines), observed in present coding genes, and by considerations involving the availability of the different kinds of amino acids. Finally, we try to explain the hydrophobicity correlation when reconstructing the events at the time of the so-called "RNA World". The whole of our investigation shows that the genetic code might be sufficiently robust to exist without the participation of pre-existing proteins, and that this robustness is a consequence of the physico-chemical properties of the four bases of the genetic system., (Copyright 2000 Academic Press.)

In many Candida species, the leucine CUG codon is decoded by a tRNA with two unusual properties: it is a ser-tRNA and, uniquely, has guanosine at position 33 (G33). Using a combination of enzymatic (V1 RNase, RnI nuclease) and chemical (Pb(2+), imidazole) probing of the native Candida albicans ser-tRNACAG, we demonstrate that the overall tertiary structure of this tRNA resembles that of a ser-tRNA rather than a leu-tRNA, except within the anticodon arm where there is considerable disruption of the anticodon stem. Using non-modified in vitro transcripts of the C. albicans ser-tRNACAG carrying G, C, U or A at position 33, we demonstrate that it is specifically a G residue at this position that induces the atypical anticodon stem structure. Further quantitative evidence for an unusual structure in the anticodon arm of the G33-tRNA is provided by the observed change in kinetics of methylation of the G at position 37, by purified Escherichia coli m(1)G37 methyltransferase. We conclude that the anticodon arm distortion, induced by a guanosine base at position 33 in the anticodon loop of this novel tRNA, results in reduced decoding ability which has facilitated the evolution of this tRNA without extinction of the species encoding it., (Copyright 1999 Academic Press.)

The recognition manner of tRNA(Leu), a class II tRNA characterized by a long variable arm, by leucyl-tRNA synthetase from an extreme halophilic archaea, Haloferax volcanii, was studied using the in vitro transcription system. It was found that the discriminator base (A73) and the long variable arm, especially the specific loop sequence A47CG47D and U47H at the base of this helix, are significant for recognition by LeuRS. An appropriate stem length of the variable arm was also required. Base substitutions in the anticodon arm did not affect the leucylation activity. Transplantation of both the discriminator base and the variable arm of tRNA(Leu) was not sufficient to introduce leucylation activity to tRNA(Ser). Insertion of an additional nucleotide into the D-loop, which is not involved in the direct interaction with LeuRS, converted tRNA(Ser) to an efficient leucine acceptor. This suggests that differences in the tertiary structure play a key role in eliminating tRNA(Ser). The sequence-specific recognition of the long variable arm of tRNA(Leu) has not been observed in any of other organisms reported, such as Escherichia coli, yeast or human. On the other hand, the mode of discrimination from non-cognate tRNAs is similar to that in E. coli in that differences in the tertiary structure play a key role. Similarity extends to the substrate stringency, exemplified by a cross-species aminoacylation study showing that no class II tRNAs from E. coli or yeast can be leucylated by H. volcanii LeuRS. Our results have implications for the understanding of the evolution of the recognition system of class II tRNA., (Copyright 1999 Academic Press.)

The tRNA anticodon loops always comprise seven nucleotides and is involved in many recognition processes with proteins and RNA fragments. We have investigated the nature and the possible interactions between the first (32) and last (38) residues of the loop on the basis of the available sequences and crystal structures. The data demonstrate the conservation of a bifurcated hydrogen bond interaction between residues 32 and 38, located at the stem/loop junction. This interaction leads to the formation of a non-canonical base-pair which is preserved in the known crystal structures of tRNA/synthetase complexes. Among the tRNA and tDNA sequences, 93 % of the 32.38 oppositions can be assigned to two families of isosteric base-pairs, one with a large (86 %) and the other with a much smaller (7 %) population. The remainder (7 %) of the oppositions have been assigned to a third family due to the lack of evidence for assigning them into the first two sets. In all families, the Y32.R38 base-pairs are not isosteric upon reversal (like the sheared G.A or wobble G.U pairs), explaining the strong conservation of a pyrimidine at position 32. Thus, the 32.38 interaction extends the sequence signature of the anticodon loop beyond the conserved U-turn at position 33 and the usually modified purine at position 37. A comparison with other loops containing both a singly hydrogen-bonded base-pair and a U-turn suggests that the 32.38 pair could be involved in the formation of a base triple with a residue in a ribosomal RNA component. It is also observed that two crystal structures of ribozymes (hammerhead and leadzyme) present similar base-pairs at the cleavage site., (Copyright 1999 Academic Press.)

Crystallographic studies of the aspartyl-tRNA synthetase-tRNA(Asp)complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNA(Asp). Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNA(Asp)recognition and acylation. The results showed that contacts with the acceptor A of tRNA(Asp)by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNA(Asp). Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state. These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis. This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site. This signal might be conveyed via tRNA(Asp)as suggested by the observed conformational change of this molecule upon interaction with AspRS. From binding free energy values corresponding to the different AspRS-tRNA(Asp)interaction domains, it might be concluded that upon complex formation, the anticodon interacts first. Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNA(Asp)and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs., (Copyright 1999 Academic Press.)

Significant departures from the canonical (cloverleaf) secondary structure of transfer (t)RNAs can be found among the mitochondrial (m)tRNAs of higher metazoans; these mtRNAs thus pose a challenge to the concept of an invariant, L-shaped tertiary conformation for all tRNAs. For bovine mtRNASer(AGY), which lacks the entire "dihydrouridine" (dhU) arm, two distinct tertiary models have been proposed: the first model preserves the L-shaped conformation at the expense of overall size; the second model preserves the absolute distance between the 3' terminus and the anticodon loop, while allowing the acceptor-anticodon interstem angle to vary. We have tested the central predictions of these two models by performing a series of transient electric birefringence measurements on bovine mtRNASer(AGY) constructs in which the aminoacyl-acceptor and anticodon stems were each extended by approximately 70 bp. This mtRNA species is particularly amenable to analysis, since the native bovine (heart) mtRNA is completely unmodified outside of the anticodon loop. For magnesium ion concentrations above 1 mM, the interstem angle for the extended mtRNA, 120(+/-5) degrees, is approximately 50% larger than the corresponding angle for yeast tRNAPhe (70-80 degrees) under the same ionic conditions. Furthermore, the interstem angles of the two tRNAs exhibit strikingly different responses to the addition of magnesium ions: the interstem angle for yeast tRNAPhe is reduced by nearly 50 % upon addition of 2 mM magnesium ions, whereas the angle for mtRNASer(AGY) increases by about 10%. Our data thus support a central prediction of the second model; namely, that truncated mtRNAs will possess more open interstem angles. In addition, we demonstrate that birefringence amplitude data can be used to provide model-independent estimates for the interstem angles., (Copyright 1999 Academic Press.)

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNALys,3. The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters derived from temperature dependent UV measurements. The pseudouridine-containing hairpin is thermodynamically more stable than the unmodified hairpin by 5 degreesC, and this corresponds with increased base stacking on the 3' side of the tRNA anticodon loop. An A+38-C32 base-pair also forms at the base of the anticodon stem with an approximate pKa of 6 for A38. Formation of the A+-C base-pair increases the Tm of both pseudouridine modified and unmodified RNA hairpins by 5-6 degreesC, and decreases the DeltaG degrees for hairpin formation by 1 kcal/mol. Solution structures were determined for both psi39 and unmodified hairpins under limiting pH conditions at pH 5 and pH 7 to assess the structural effects of both psi modification and the additional A+-C base-pair on tRNALys,3 structure. The A+38-C32 base-pair strengthens the 31-39 base-pair, and induces formation of a dynamic U33-A37 base-pair that effectively reduces the normal seven nucleotide anticodon loop to a three nucleotide UUU loop. These undermodified tRNALys,3 anticodon loops are distinctly different from those seen for other tRNAs exemplified by tRNAPhe. The conformation of the tRNA loop has important implications for the role of nucleoside modification in codon-anticodon recognition and for utilization of tRNALys,3 by HIV-1 as the native reverse transcriptase primer., (Copyright 1999 Academic Press.)

The origin of the organisation of the genetic code reflects both the biosynthetic relationships between amino acids and the physicochemical interactions between these and anticodons; moreover, these two forces do not act independently. It must therefore be explained why it is simultaneously true that the anticodons of product amino acids were assigned prior to their biosynthetic appearance and that physicochemical correlations must exist between anticodons and amino acids. This gives rise to difficulties of interpretation, even of a more general nature, in the theories that have been proposed to explain the origin of the genetic code; a hypothesis is thus presented in this manuscript. In particular, the hypothesis suggests that RNA hairpin structures, the ancestors of tRNAs, housing anticodon-like nucleotides in the stem were charged with precursor amino acids. As the precursor amino acids gradually developed into product amino acids, a coevolution came into being between the development of amino acids and that of anticodons, with a concomitant formation of the complete tRNA molecule thought direct duplication of the hairpin structures. All this led to the definition of the genetic code organisation. Furthermore, this made it possible for the evolving anticodons to select the emerging product amino acids, which the hypothesis therefore considers to be unspecified in the initial phase of genetic code evolution but which were selected also because of their ability to interact with anticodons. In this way the main obstacle to interpretation is removed. Fossils of these events can now be observed in some amino acid-modified nucleosides specifically located in the tRNA anticodon loops. This is all presented in the framework of a general discussion of the ideas and data in favour of a late origin of the genetic code, as opposed to an early origin in the context of the theories proposed to explain the origin of the genetic code organisation.

The hydration of the tRNA(Asp) anticodon hairpin was investigated through the analysis of six 500 ps multiple molecular dynamics (MMD) trajectories generated by using the particle mesh Ewald method for the treatment of the long-range electrostatic interactions. Although similar in their dynamical characteristics, these six trajectories display different local hydration patterns reflecting the landscape of the "theoretical" conformational space being explored. The statistical view gained through the MMD strategy allowed us to characterize the hydration patterns around important RNA structural motifs such as a G-U base-pair, the anticodon U-turn, and two modified bases: pseudouridine and 1-methylguanine. The binding of ammonium counterions to the hairpin has also been investigated. No long-lived hydrogen bond between water and a 2'-hydroxyl has been observed. Water molecules with long-residence times are found bridging adjacent pro-Rp phosphate atoms. The conformation of the pseudouridine is stiffened by a water-mediated base-backbone interaction and the 1-methylguanine is additionally stabilized by long-lived hydration patterns. Such long-lived hydration patterns are essential to ensure the structural integrity of this hairpin motif. Consequently, our simulations confirm the conclusion reached from an analysis of X-ray crystal structures according to which water molecules form an integral part of nucleic acid structure. The fact that the same conclusion is reached from a static and a dynamic point of view suggests that RNA and water together constitute the biologically relevant functional entity.

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.

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.

In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coli it is present only in tRNAArg. The corresponding tRNA genes all have adenosine at position 34. Using as substrates in vitro T7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34. The homologous E. coli enzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG. The base conversion occurs by a hydrolytic deamination-type reaction. This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectrometry analysis of the reaction product after in vitro modification in [18O]water. This newly characterised tRNA:adenosine 34 deaminase was partially purified from yeast. It has a molecular mass of approximately 75 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA. The observed dependence of the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture. Enzymatic recognition of tRNA does not depend on the presence of any "identify" nucleoside other than adenosine 34. Likewise, the presence of pseudouridine 32 or 1-methyl-guanosine 37 in the anticodon loop does not interfere with inosine 34 biosynthesis. However, the efficacy of adenosine 34 to inosine 34 conversion depends on the nucleotide sequence of the anticodon loop and its proximal stem, the best tRNA substrates being those with a purine at position 35. Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tRNA to be substrate for the yeast tRNA:adenosine 34 deaminase. Evidently, the activity of yeast tRNA:adenosine 34 deaminase depends more on the global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular nucleotide other than adenosine 34. An apparent K(m) of 2.3 nM for its natural substrate tRNASer (anticodon AGA) was measured. Altogether, these results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast.

Computer graphics simulation of the interaction between the codon-anticodon duplexes containing adenine in the first (wobble) position of the anticodons, and bound to the ribosomal A- and P-sites, was made. This demonstrated that widespread use of adenine in the wobble position in anticodons should lead to a low efficiency of ribosomal translation, since the wobble A of the P-site tRNA weakens the codon-dependent binding of aminoacyl-tRNA at the A-site via interduplex interaction. Besides the canonical partner U, the wobble A of aminoacyl-tRNA can recognize A, C, G in the third position of the codon by the formation of the propeller twist in the wobble pairs AA, AC, AG. The conversion of the wobble A into inosine improves its pairing with the codon bases (the pairs IA and IC, unlike AA and AC, should not form the propeller twist leading to the deformation of base-base hydrogen bonds) and should reduce an adverse effect of the P-site wobble adenine on the formation of the A-site duplex. The consequence of the interaction between the ribosomal P- and E-site duplexes has been formulated. According to this the E-site wobble A should enhance the probability of frameshifting. These properties of the wobble A and I could be a reason why A is very rarely observed in the first anticodon position and why evolutionary processes have developed the enzyme which modifies the wobble A to I. The results obtained can be subjected to direct experimental tests.

Wobble rules for modified residues in the first anticodon position are derived. All known modifications are considered individually. Stereochemical analysis was made taking into account the interaction between the ribosomal A and P-site bound codon-anticodon duplexes. The wobble base-pair was considered as the right one if its formation did not lead to an uncompensated loss of hydrogen bonds or polar atom-ion bonds. From this requirement it follows that all modifications of U should restrict its translational specificity to purines (with the exception of xo5U, which should decode A, G and U). The restriction is carried out in a unified way: modifications inhibit the large propeller twist resulting from an increase of about 35 degrees in the torsion angle of the anticodon wobble base, interacting with the third codon base via a hydrogen-bonded water molecule. Such a twist is required to avoid a loss of the hydrogen bond of the bonded water molecule. The modifications in S2U, Se2U and Um should weaken their pairing with G, because they deform one of the two hydrogen bonds of the guanine NH2 group. G should be recognized by Se2U better than by S2U for the reason that the hydrogen bond Se...HN is weaker than the hydrogen bond S...HN. Among the modifications of C and G only that in k2C has a pronounced effect on wobble. The nucleoside k2C should pair only with A. The N-2 atom of k2C should be in the pyramidal state. The consequences following from the interduplex interaction are formulated. According to one of them, adenosine in the wobble position of the P-site tRNA should destabilize the A-site duplex. This can serve as an explanation for the fact that adenosine is very rarely observed in the anticodon wobble position.

We propose three-dimensional models for animal mitochondrial (amt) tRNAs lacking the D-domain based on consideration of universal constraints on tRNA to maintain functionality. The available tRNA sequences are classified into two groups, and distinct models are proposed for both classes derived from common structural features. The distance between the anticodon and the acceptor stem is comparable in the models and corresponds to that observed in conventional tRNAs. This fact averts the problem of how a shorter mitochondrial tRNA could function within the context of a protein synthesis machinery suited to full-sized tRNAs. In the models, the angle which defines the relationship between the helical domains composed of the acceptor/T-stem and the anticodon/D-stem is greater than in conventional tRNAs. These structures resemble more a "boomerang" than an "L". However, even in the boomerang model, the inner surface of tRNA would be sufficiently uncluttered to avoid steric clashes when two tRNA molecules cohabit the ribosome.

Molecular recognition of Escherichia coli tRNA(Ile) by the cognate isoleucyl-tRNA synthetase (IleRS) was studied by analyses of chemical footprinting with N-nitroso-N-ethylurea and aminoacylation kinetics of variant tRNA(Ile) transcripts prepared with bacteriophage T7 RNA polymerase. IleRS binds to the acceptor, dihydrouridine (D), and anticodon stems as well as to the anticodon loop. The "complete set" of determinants for the tRNA(Ile) identity consists of most of the nucleotides in the anticodon loop (G34, A35, U36, t6A37 and A38), the discriminator nucleotide (A73), and the base-pairs in the middle of the anticodon, D and acceptor stems (C29.G41, U12.A23 and C4.G69, respectively). As for the tertiary base-pairs, two are indispensable for the isoleucylation activity, whereas the others are dispensable. Correspondingly, some of the phosphate groups of these dispensable tertiary base-pair residues were shown to be exposed to N-nitroso-N-ethylurea when tRNA(Ile) was bound with IleRS. Furthermore, deletion of the T psi C-arm only slightly impaired the tRNA(Ile) activity. Thus, it is proposed that the recognition by IleRS of all the widely distributed identity determinants is coupled with a global conformational change that involves the loosening of a particular set of tertiary base-pairs of tRNA(Ile).

The number and relative amount of isoacceptor tRNAs for each amino acid in Micrococcus luteus, a Gram-positive bacterium with high genomic G + C content, have been determined by sequencing their anticodon loop and its adjacent regions and by selective labelling of tRNAs. Thirty-one tRNA species with 29 different anticodon sequences have been detected. All the tRNAs have G or C at the anticodon first position except for tRNA(ICGArg) and tRNA(NGASer), in response to the abundant usage of NNC and NNG codons. No tRNA with the anticodon UNN capable of translating codon NNA has been detected, in accordance with a very low or zero usage of NNA codons. The relative amount of isoacceptor tRNAs for an amino acid determined by selective labelling strongly correlates with usage of the corresponding codons. On the basis of these and other observations in this and other eubacterial species, we conclude that the relative amount and anticodon composition of isoacceptor tRNA species are flexible, and their changes are mainly adaptive phenomena that have been primarily affected by codon usage, which in turn is affected by directional mutation pressure.