Effect of uridine dethiolation in the anticodon triplet of tRNA(Glu) on its association with tRNA(Phe)

The effect of U(34) dethiolation on the anticodon-anticodon association between E. coli tRNA(Glu) and yeast tRNA(Phe) has been studied by the temperature jump relaxation technique. An important destabilization upon replacement of the thioketo group of s2U(34) by a keto group, was revealed by a lowering of melting temperature of about 20 degrees C. The measured kinetic parameters indicated that this destabilization effect was originated in an increase of dissociation and a decrease of association rate constants by a factor of 4 to 5. Modifications in both stacking interactions and flexibility in the anticodon loop would be responsible for this effect.     

The wobble hypothesis revisited: uridine-5-oxyacetic acid is critical for reading of G-ending codons

According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo⁵U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo⁵U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo⁵U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo⁵U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo⁵U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo⁵U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34.     

Structural Studies of 2-Thiouridine in RNA

Abstract

A pentamer RNA sequence, Gs²UUUC, and a s²U containing 14-mer RNA tetraloop hairpin were synthesized and characterized by NMR and by UV melting studies. These oligonucleotides were used as models to understand the effect of 2-thiouridine substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through sU-34 modification. The magnitude of the effect of sU in our model system is comparable to the 20° C stabilization reported for 2-thiolation in a codonanticodon model system composed of two tRNAs with complementary anticodon sequences.     

Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA₂^Ile to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNA^Ile-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA₂^Ile is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA₁^Ile, in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.     

Identification and codon reading properties of 5-cyanomethyl uridine,a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA₂^Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA₂^Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.     

The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)

The conserved U54 in tRNA is often modified to 5-methyluridine (m⁵U) and forms a reverse Hoogsteen base pair with A58 that stabilizes the L-shaped tRNA structure. In Gram-positive and some Gram-negative eubacteria, m⁵U54 is produced by folate/FAD-dependent tRNA (m⁵U54) methyltransferase (TrmFO). TrmFO utilizes N⁵,N¹⁰-methylenetetrahydrofolate (CH₂THF) as a methyl donor. We previously reported an in vitro TrmFO assay system, in which unstable [¹⁴C]CH₂THF was supplied from [¹⁴C]serine and tetrahydrofolate by serine hydroxymethyltransferase. In the current study, we have improved the TrmFO assay system by optimization of enzyme and substrate concentrations and introduction of a filter assay system. Using this assay, we have focused on the tRNA recognition mechanism of TrmFO. 42 tRNA mutant variants were prepared, and experiments with truncated tRNA and microhelix RNAs revealed that the minimum requirement of TrmFO exists in the T-arm structure. The positive determinants for TrmFO were found to be the U54U55C56 sequence and G53-C61 base pair. The gel mobility shift assay and fluorescence quenching showed that the affinity of TrmFO for tRNA in the initial binding process is weak. The inhibition experiments showed that the methylated tRNA is released before the structural change process. Furthermore, we found that A38 prevents incorrect methylation of U32 in the anticodon loop. Moreover, the m¹A58 modification clearly accelerates the TrmFO reaction, suggesting a synergistic effect of the m⁵U54, m¹A58, and s²U54 modifications on m⁵s²U54 formation in Thermus thermophilus cells. The docking model of TrmFO and the T-arm showed that the G53-C61 base pair is not able to directly contact the enzyme.     

Inhibition and Substrate Competition Kinetics in Analysis of Porcine Thyroid Alkaline Ribonuclease's Specificity Toward Synthetic Rna's and Trna

Abstract

Inhibition and substrate competition kinetics demonstrated that tRNA is a highly preferred substrate of thyroid alkaline RNase. The pyrimidine-specific RNase cleaved poly(C) 2.8 × 10⁵ faster than poly(U). k_cat: K_M ratios for tRNA and poly(C) based on molecular weights failed to predict preference when both were present. Competition experiments between poly(C) and tRNA revealed tRNA was a tight-binding competing substrate and the cytidylate residues in the 3prime;-CCA terminus of tRNA were preferred about 280: 1 over those in poly(C). Poly(U) was competitive with tRNA. When poly(C) was the substrate, inhibition type by poly(G) depended on poly(G) concentration. Neither tRNA lacking its 3prime; terminal cytidylyl(3prime;-5prime;)adenosine and terminating in a 2prime;:3prime; cCMP residue, tRNA lacking its 3prime; terminal 5prime;AMP residue, guanosine, nor guanylyl(3prime;-5prime;)guanylyl(3prime;-5prime;)guanosine were inhibitors. Product inhibition by adenosine and 2prime;:3prime; cCMP showed the kinetic mechanism for cleavage of tRNA was ordered uni bi.     

A new kinetic model reveals the synergistic effect of E-, P- and A-sites on +1 ribosomal frameshifting

Programmed ribosomal frameshifting (PRF) is a process by which ribosomes produce two different polypeptides from the same mRNA. In this study, we propose three different kinetic models of +1 PRF, incorporating the effects of the ribosomal E-, P- and A-sites toward promoting efficient +1 frameshifting in Escherichia coli. Specifically, the timing of E-site tRNA dissociation is discussed within the context of the kinetic proofreading mechanism of aminoacylated tRNA (aa-tRNA) selection. Mathematical modeling using previously determined kinetic rate constants reveals that destabilization of deacylated tRNA in the E-site, rearrangement of peptidyl-tRNA in the P-site, and availability of cognate aa-tRNA corresponding to the A-site act synergistically to promote efficient +1 PRF. The effect of E-site codon:anticodon interactions on +1 PRF was also experimentally examined with a dual fluorescence reporter construct. The combination of predictive modeling and empirical testing allowed the rate constant for P-site tRNA slippage (k_s) to be estimated as k_s ≈1.9 s⁻¹ for the release factor 2 (RF2) frameshifting sequence. These analyses suggest that P-site tRNA slippage is the driving force for +1 ribosomal frameshifting while the presence of a ‘hungry codon’ in the A-site and destabilization in the E-site further enhance +1 PRF in E. coli.     

Distribution of Cytokinin-active Ribonucleosides in Wheat Germ tRNA Species

The distribution of cytokinin activity in wheat (Triticum aestivum) germ tRNA fractionated by BD-cellulose and RPC-5 chromatography has been examined. As in other organisms, the cytokinin moieties in wheat germ tRNA appear to be restricted to tRNA species that would be expected to respond to codons beginning with U. Only a few of the wheat germ tRNA species in this coding group actually contain cytokinin modifications. Cytokinin activity was associated with isoaccepting tRNA^Ser species and with a minor tRNA^Leu species from wheat germ. All other wheat germ tRNA species corresponding to codons beginning with U were devoid of cytokinin activity in the tobacco callus bioassay.     

Position 34 of tRNA is a discriminative element for m5C38 modification by human DNMT2

Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNA^Asp(GUC), tRNA^Gly(GCC) and tRNA^Val(AAC). Intriguingly, for tRNA^Asp(GUC) and tRNA^Gly(GCC), G34 is the discriminator element; whereas for tRNA^Val(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C³²U³³(G/I)³⁴N³⁵ (C/U)³⁶A³⁷C³⁸ motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism.     

Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA – A Possible Intermediate in Its Selenation?

The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U) in tRNA specific for Gln. The sulfur of (c)mnm⁵s²U may be exchanged by selenium (Se)–a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C₁₀H₁₇-fragment) attached to the s² group of mnm⁵s²U and of cmnm⁵s²U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm⁵ges²U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH⁺ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm⁵s²U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA.     

Undermodification in the first position of the anticodon of supG-tRNA reduces translational efficiency

Summary Two mutants of Escherichia coli, trmC1 and trmC2, which are both defective in the synthesis of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) were utilized to study the function of this complex modified nucleoside. Transfer RNAs specific for glutamine, glutamic acid and lysine as well as a specific ochre suppressor derived from lysine tRNA (tRNA _UAA ^lys encoded by the supG allele), contain this modified nucleoside at position 34 (the wobble position). It was found that two different undermodified derivatives of mnm⁵s²U were present in the two trmC mutants, which suggests that the two mutations affect two different enzymatic activities. Using the lacI-Z fusion system (Miller and Albertini 1983), we found that the efficiency of supG-mediated suppression was reduced to 30%–90% of the wild-type value in the trmC mutants. The modificationdeficient supG-tRNA in the mutants showed a higher sensitivity to codon context than the normal tRNA _UAA ^lys .     

Functional importance of Ψ38 and Ψ39 in distinct tRNAs,amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U₃₈ and U₃₉ in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ₃₈ or Ψ₃₉ is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA^Gln(UUG), which normally has Ψ₃₈. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s²U moiety of mcm⁵s²U₃₄ of tRNA^Gln(UUG) and the other two cytoplasmic species with mcm⁵s²U₃₄, that the reduced s²U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s²U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ_38,39 provides evidence that Ψ₃₈ is important for function of tRNA^Gln(UUG) at permissive temperature, and indicates that Ψ₃₉ is important for the function of tRNA^Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA^Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ₃₈ and Ψ₃₉ in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.     

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

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

The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes

In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), 5-methoxycarbonylmethyluridine (mcm⁵U), 5-carbamoylmethyluridine (ncm⁵U) or 5-carbamoylmethyl-2′-O-methyluridine (ncm⁵Um) nucleosides in the anticodon at the wobble position (U₃₄). Earlier we showed that mutants unable to form the side chain at position 5 (ncm⁵ or mcm⁵) or lacking sulphur at position 2 (s²) of U₃₄ result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm⁵ and s² side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors.     

Contribution of ribosomal residues to P-site tRNA binding

Structural studies have revealed multiple contacts between the ribosomal P site and tRNA, but how these contacts contribute to P-tRNA binding remains unclear. In this study, the effects of ribosomal mutations on the dissociation rate (k_off) of various tRNAs from the P site were measured. Mutation of the 30S P site destabilized tRNAs to various degrees, depending on the mutation and the species of tRNA. These data support the idea that ribosome-tRNA interactions are idiosyncratically tuned to ensure stable binding of all tRNA species. Unlike deacylated elongator tRNAs, N-acetyl-aminoacyl-tRNAs and tRNA^fMet dissociated from the P site at a similar low rate, even in the presence of various P-site mutations. These data provide evidence for a stability threshold for P-tRNA binding and suggest that ribosome-tRNA^fMet interactions are uniquely tuned for tight binding. The effects of 16S rRNA mutation G1338U were suppressed by 50S E-site mutation C2394A, suggesting that G1338 is particularly important for stabilizing tRNA in the P/E site. Finally, mutation C2394A or the presence of an N-acetyl-aminoacyl group slowed the association rate (k_on) of tRNA dramatically, suggesting that deacylated tRNA binds the P site of the ribosome via the E site.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA^Cys, tRNA^Trp, tRNA^Tyr, a major peak of tRNA^Phe, and a large fraction of tRNA^Leu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA^Ser species and with minor tRNA^Leu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.