Lack of pseudouridine 38/39 in the anticodon arm of yeast cytoplasmic tRNA decreases in vivo recoding efficiency

Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.     

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 .     

Systematic alterations in the anticodon arm make tRNA(Glu)-Suoc a more efficient suppressor.

Using site-specific mutagenesis, we constructed five more efficient variants of tRNA(Glu)-Suoc, an extremely inefficient ochre suppressor. Each variant has an extended anticodon, or region of the anticodon arm, which is more similar to that found in normal tRNAs which translate codons Uxx. Suppressor efficiency invariably increases with similarity of the extended anticodon to that of a normal Uxx-translating tRNA. Altered nucleotides in both helix and loop strongly affect efficiency, with no position dependence and no significant interaction between substitutions. The variant with all substitutions is 230-fold more efficient (in one context) than the parental tRNA(Glu)-Suoc. Two other unexpected variants seem to be 'context mutants', having altered response to message context.     

The anticodon of the maize chloroplast gene for tRNA Leu UAA is split by a large intron.

The maize chloroplast gene encoding tRNA Leu UAA has been sequenced. It contains a 458 base pair intron between the first and second bases of the anticodon. The tRNA is 88 nucleotides long (the 3'-terminal CCA sequence included which, however, is not encoded by the gene) and differs in only four nucleotides (modified nucleotides are not considered) from the corresponding isoacceptor from bean chloroplasts. The unusual position of the intron in this maize chloroplast tRNA gene suggests a splicing model different from that generally accepted for eukaryotic split tRNA genes.     

Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm

Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome. To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe. The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation. Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe. Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19. Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material. Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.     

Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.     

Translocation of a tRNA with an extended anticodon through the ribosome

Coordinated translocation of the tRNA-mRNA complex by the ribosome occurs in a precise, stepwise movement corresponding to a distance of three nucleotides along the mRNA. Frameshift suppressor tRNAs generally contain an extra nucleotide in the anticodon loop and they subvert the normal mechanisms used by the ribosome for frame maintenance. The mechanism by which suppressor tRNAs traverse the ribosome during translocation is poorly understood. Here, we demonstrate translocation of a tRNA by four nucleotides from the A site to the P site, and from the P site to the E site. We show that translocation of a punctuated mRNA is possible with an extra, unpaired nucleotide between codons. Interestingly, the NMR structure of the four nucleotide anticodon stem-loop reveals a conformation different from the canonical tRNA structure. Flexibility within the loop may allow conformational adjustment upon A site binding and for interacting with the four nucleotide codon in order to shift the mRNA reading frame.     

Interaction of yeast tRNA(Phe) anticodon arm with 40S ribosomal subunit of rabbit liver.

The 15-nucleotide analog of yeast tRNA(Phe) anticodon arm binds cooperatively to two sites of poly(U) programmed 40S ribosome like intact tRNA(Phe). The cooperativity coefficients appeared to be about 4 for tRNA(Phe) and 50 for its anticodon arm. Anticodon arm contributes the majority of free energy of tRNA binding to a programmed 40S ribosomal subunit. The correct codon-anticodon pairing seems to play the key role in the cooperativity origin. Contrary to the anticodon arm template independent binding of the whole tRNA to the small ribosomal subunit is revealed.     

Role of the anticodon in recognition of tRNA by aminoacyl-tRNA-synthetases

The nucleic acid-protein interactions are considered for mutual recognition of transfer RNAs and cognate aminoacyl-tRNA synthetases. The validity of the first recognition hypothesis (1964) which postulated the participation of anticodons in this process has been proved both for prokaryote and eukaryote tRNA species. This conclusion stems from numerous data obtained by means of different methodological approaches. These experimental observations concern the majority of tRNAs belonging to the main structural class of tRNAs with a short variable loop. Structural basis of recognition for some tRNAs (with long variable loop) remains obscure.     

The three conformations of the anticodon loop of yeast tRNA(Phe)

The complex conformational states of the anticodon loop of yeast tRNA(Phe) which we had previously studied with relaxation experiments by monitoring fluorescence of the naturally occurring Wye base, are analyzed using time and polarization resolved fluorescence measurements at varying counterion concentrations. Synchrotron radiation served as excitation for these experiments, which were analyzed using modulating functions and global methods. Three conformations of the anticodon loop are detected, all three occurring in a wide range of counterion concentrations with and without Mg2+, each being identified by its typical lifetime. The fluorescence changes brought about by varying the ion concentrations, previously monitored by steady state fluorimetry and relaxation methods, are changes in the population of these three conformational states, in the sense of an allosteric model, where the effectors are the three ions Mg2+, Na+ and H+. The population of the highly fluorescent M conformer (8ns), most affine to magnesium, is thus enhanced by that ligand, while the total fluorescence decreases as lower pH favors the H+-affine H conformer (0.6ns). Na+-binding of the N conformer (4ns) is responsible for complex fluorescence changes. By iterative simulation of this allosteric model the equilibrium and binding constants are determined. In turn, using these constants to simulate equilibrium fluorescence titrations reproduces the published results.     

Starvation-induced cleavage of the tRNA anticodon loop in Tetrahymena thermophila

Amino acid deprivation triggers dramatic physiological responses in all organisms, altering both the synthesis and destruction of RNA and protein. Here we describe, using the ciliate Tetrahymena thermophila, a previously unidentified response to amino acid deprivation in which mature transfer RNA (tRNA) is cleaved in the anticodon loop. We observed that anticodon loop cleavage affects a small fraction of most or all tRNA sequences. Accumulation of cleaved tRNA is temporally coordinated with the morphological and metabolic changes of adaptation to starvation. The starvation-induced endonucleolytic cleavage activity targets tRNAs that have undergone maturation by 5' and 3' end processing and base modification. Curiously, the majority of cleaved tRNAs lack the 3' terminal CCA nucleotides required for aminoacylation. Starvation-induced tRNA cleavage is inhibited in the presence of essential amino acids, independent of the persistence of other starvation-induced responses. Our findings suggest that anticodon loop cleavage may reduce the accumulation of uncharged tRNAs as part of a specific response induced by amino acid starvation.     

A major lysine tRNA with a CUU anticodon in insect mitochondria

We have sequenced a lysine tRNA from mosquito mitochondria that has the anticodon CUU. The preponderance of AAA lysine codons in insect mitochondrial genes, the parsimonious organization of the genomes, and the fact that this tRNA is a major component of the mosquito mitochondrial tRNA complement, lead us to suggest that the CUU anticodon recognizes AAC and AAA codons.     

The bases of the tRNA anticodon loop are independent by genetic criteria.

We employed two methods to study the translational role of interactions between anticodon loop nucleotides. Starting with a set of previously constructed weakly-suppressing anticodon loop mutants of Su7, we searched for second-site revertants that increase amber suppressor efficiency. Though hundreds of revertants were characterized, no second-site revertants were found in the anticodon loop. Second site reversion was detected in the D-stem, thereby demonstrating the efficacy of the search method. As a second method for detecting interactions, we used site-directed mutagenesis to construct multiple mutations in the anticodon loop. These multiple mutants are very weak suppressors and have translational activities that are equal to or lower than that predicted for the independent action of single mutations. We conclude that although the anticodon loop sequence of Su7 has an optimal structure for the translation of amber codons, we find no evidence that interactions between loop bases can enhance translational efficiency.     

A modified nucleoside located in the anticodon of proline tRNA from Torulopsis utilis is 5-carbamoylmethyluridine

A modified nucleoside has been isolated from the first position of the anticodon of Torulopsis utilis tRNAPro. It was identified to be an uridine derivative, 5-carbamoylmethyluridine from analyses of its UV, 1H-NMR, and secondary ion mass spectra.