A novel type of + 1 frameshift suppressor: a base substitution in the anticodon stem of a yeast mitochondrial serine-tRNA causes frameshift suppression.

We have identified a spontaneous mitochondrial mutation, mfs-1 (mitochondrial frameshift suppressor-1), which suppresses a + 1 frameshift mutation localized in the yeast mitochondrial oxi1 gene. The suppressor strain exhibits a single base change (C to U) at position 42 of the mitochondrial serine-tRNA (UCN). To our knowledge, this is the first reported case showing that a mutation in the anticodon stem of a tRNA can cause frameshift suppression. The expression and aminoacylation of the mutant tRNASer(UCN) are not significantly affected. However, the base change at position 42 has two effects: first, residue U27 of the mutant tRNA is not modified to pseudouridine as observed in wild-type tRNASer(UCN). Second, the base change and/or the lack of modification of U27 leads to an alteration in the secondary/tertiary structure of the mutant tRNA. It is possible that there are such structural changes in the anticodon loop that enable the tRNA to read a four base codon, UCCA, thus restoring the wild-type reading frame.     

Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA.

In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal; few shifts in the 19F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29.A41 base-pair in the anticodon stem with C29.G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA. Nucleotide substitution at the 3' end of the anticodon (position 36) reduced the aminoacylation efficiency (Vmax/Km) of tRNAVal by three orders of magnitude. Base replacement at the 5' end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency. Substitution of the FU29.A41 base-pair increased the Km value 20-fold, while Vmax remained almost unchanged. The FU4.A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E. coli tRNAVal, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.     

Identification of potential amino acid residues supporting anticodon recognition in yeast methionyl-tRNA synthetase

Sequence comparisons among methionyl-tRNA synthetases from different organisms reveal only one block of homology beyond the last beta strand of the mononucleotide fold. We have introduced a series of semi-conservative amino acid replacements in the conserved motif of yeast methionyl-tRNA synthetase. The results indicate that replacements of two polar residues (Asn584 and Arg588) affected specifically the aminoacylation reaction. The location of these residues in the tertiary structure of the enzyme is compatible with a direct interaction of the amino acid side-chains with the tRNA anticodon.     

Construction,aminoacylation and 80 S ribosomal complex formation with a yeast initiator tRNA having an arginine CCU anticodon

The digestion of yeast initiator methionine tRNA with mung bean nuclease and U₂ ribonuclease yielded 5'- and 3'-fragments, respectively. These two fragments together represent the entire tRNA sequence except for A₃₅, the central nucleotide of the anticodon, and the CCA terminus. Using RNA ligase, a cytosine was added and the anticodon loop having a C₃₅ was reformed. Subsequent treatment of this product with CCA-transferase yielded a full-length methionine tRNA having an arginine CCU anticodon. This recombinant tRNA^Met (CCU) was charged with methionine by the yeast tRNA synthetase. Aminoacylation of the recombinant was however less extensive than in the case of native tRNA^Met (CAU). After aminoacylation the recombinant tRNA formed an 80 S ribosomal complex.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli.

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo. We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo. For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA. Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation. The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site. The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.     

Efficient aminoacylation of tRNA(Lys,3) by human lysyl-tRNA synthetase is dependent on covalent continuity between the acceptor stem and the anticodon domain

In this work, we probe the role of the anticodon in tRNA recognition by human lysyl-tRNA synthetase (hLysRS). Large decreases in aminoacylation efficiency are observed upon mutagenesis of anticodon positions U35 and U36 of human tRNA(Lys,3). A minihelix derived from the acceptor-TPsiC stem-loop domain of human tRNA(Lys,3)was not specifically aminoacylated by the human enzyme. The presence of an anticodon-derived stem-loop failed to stimulate aminoacylation of the minihelix. Thus, covalent continuity between the acceptor stem and anticodon domains appears to be an important requirement for efficient charging by hLysRS. To further examine the mechanism of communication between the critical anticodon recognition elements and the catalytic site, a two piece semi-synthetic tRNA(Lys, 3)construct was used. The wild-type semi-synthetic tRNA contained a break in the phosphodiester backbone in the D loop and was an efficient substrate for hLysRS. In contrast, a truncated variant that lacked nucleotides 8-17 in the D stem-loop displayedseverely reduced catalytic efficiency. The elimination of key tRNA tertiary structural elements has little effect on anticodon-dependent substrate binding but severely impacts formation of the proper transition state for catalysis. Taken together, our studies provide new insights into human tRNA structural requirements for effective transmission of the anticodon recognition signal to the distal acceptor stem domain.     

Anticodon-dependent conservation of bacterial tRNA gene sequences

The residues in tRNA that account for its tertiary fold and for its specific aminoacylation are well understood. In contrast, relatively little is known about the residues in tRNA that dictate its ability to transit the different sites of the ribosome. Yet protein synthesis cannot occur unless tRNA properly engages with the ribosome. This study analyzes tRNA gene sequences from 145 fully sequenced bacterial genomes. Grouping the sequences according to the anticodon triplet reveals that many residues in tRNA, including some that are distal to the anticodon loop, are conserved in an anticodon-dependent manner. These residues evade detection when tRNA genes are grouped according to amino acid family. The conserved residues include those at positions 32, 38, and 37 of the anticodon loop, which are already known to influence tRNA translational performance. Therefore, it seems likely that the newly detected anticodon-associated residues also influence tRNA performance on the ribosome. Remarkably, tRNA genes that belong to the same amino acid family and therefore share identical residues at the second and third anticodon positions have diverged, during bacterial evolution, into highly conserved groups that are defined by the residue at the first (wobble) anticodon position. Current ideas about the properties of tRNA and the translation mechanism do not fully account for this phenomenon. The results of the present study provide a foundation for studying the adaptation of individual tRNAs to the translation machinery and for future studies of the translation mechanism.     

Transfer RNA splicing in Saccharomyces cerevisiae. Secondary and tertiary structures of the substrates

Secondary and tertiary structures of four yeast tRNA precursors that contain introns have been elucidated using limited digestion with a variety of single-strand- and double-strand-specific nucleases. The pre-tRNAs, representing the variety of intron sizes and potential structures, were: pre-tRNALeuCAA, pre-tRNALeuUAG, pre-tRNAIleUAU, and pre-tRNAPro-4UGG. Conventional tRNA cloverleaf structure is maintained in these precursors except that the anticodon loop is interrupted by the intron. The intron contains a sequence which is complementary to a portion of the anticodon loop and allows the formation of a double helix often extending the anticodon stem. The 5' and 3' splicing cleavage sites are located at either end of this helix and are single-stranded. The intron is the most sensitive region to nuclease cleavage, suggesting that it is on the surface of the molecule and available for interaction with the splicing endonuclease. Absence of Mg2+ or spermidine renders the dihydrouridine and T psi C loops of these precursors highly sensitive to nuclease digestion. These ionic effects mimic those observed for tRNAPhe and suggest that the tRNA portion of these precursors has native tRNA structure. We propose consensus secondary and tertiary structures which may be of significance to eventual understanding of the mechanism of yeast tRNA splicing.     

tRNA prefers to kiss.

Six RNA aptamers that bind to yeast phenylalanine tRNA were identified by in vitro selection from a random-sequence pool. The two most abundantly represented aptamers interact with the tRNA anticodon loop, each through a sequence block with perfect Watson-Crick complementarity to the loop. It was possible to truncate one of these aptamers to a simple hairpin loop that forms a classical 'kissing complex' with the anticodon loop. Three other aptamers have nearly complete complementarity to the anticodon loop. The sixth aptamer has two sequence blocks, one complementary to the tRNA T loop and the other to the D loop; this aptamer binds better to a mutant tRNA that disrupts the normal D-loop/T-loop tertiary interaction than to the wild-type tRNA. Selection of complements to tRNA loops occurred despite an attempt to direct binding to tertiary structural features of tRNA. This serves as a reminder of how special the RNA-RNA interactions are that are not based on complementarity. Nonetheless, these aptamers must present the tRNA complement in some special structural context; the simple single-strand complement of the anticodon loop did not bind tRNA effectively.     

Efficient aminoacylation of resected RNA helices by class II aspartyl-tRNA synthetase dependent on a single nucleotide.

We show here that small RNA helices which recapitulate part or all of the acceptor stem of yeast aspartate tRNA are efficiently aminoacylated by cognate class II aspartyl-tRNA synthetase. Aminoacylation is strongly dependent on the presence of the single-stranded G73 'discriminator' identity nucleotide and is essentially insensitive to the sequence of the helical region. Substrates which contain as few as 3 bp fused to G73CCAOH are aspartylated. Their charging is insensitive to the sequence of the loop closing the short helical domains. Aminoacylation of the aspartate mini-helix is not stimulated by a hairpin helix mimicking the anticodon domain and containing the three major anticodon identity nucleotides. A thermodynamic analysis demonstrates that enzyme interactions with G73 in the resected RNA substrates and in the whole tRNA are the same. Thus, if the resected RNA molecules resemble in some way the earliest substrates for aminoacylation with aspartate, then the contemporary tRNA(Asp) has quantitatively retained the influence of the major signal for aminoacylation in these substrates.