Serine-inserting UAA suppression mediated by yeast tRNASer

The number of loci that give rise to serine-inserting UAA suppressors in the yeast Saccharomyces cerevisiae was determined by examining over 100 of the revertants that suppressed the two UAA markers his4-1176 and leu2-1: the his4-1176 marker is suppressed by serine-inserting but not by tyrosine- or leueine-inserting suppressors and the leu2-1 marker is suppressed by all UAA suppressors. The suppressors could be assigned to one or other of the four loci: SUP16 and SUP17. which were previously known to yield serine-inserting suppressors, and SUP19 and SUP22. The chromosomal map position of SUP19 suggested that it may be allelic to the previously reported suppressor SUP20, while the SUP22 suppressor has not been described. Representatives of all of the four suppressors were found to insert serine at the UAA site in iso-1-cytochrome c from suppressed cyc1-72 strains. The degree of suppression by the serine-inserting suppressors was SUP16 > SUP17 > SUP19 > SUP22. The efficiency of suppression of each of the four serine suppressors was increased by the chromosomal mutation sal and by the cytoplasmic determinant ψ⁺. Read-through of the synthetase gene of the RNA bacteriophage Qβ in a cell-free system was used to demonstrate that tRNA^Ser from SUP16, SUP17 and SUP19 strains can translate UAA codons. In contrast, tRNA^Ser or total tRNA from SUP22 strains had no suppressing activity. The results suggest that the three loci SUP16, SUP17 and SUP19 encode iso-accepting species of tRNA^Ser, and that the UAA suppression is mediated by mutationally altered tRNA molecules. The mechanism of SUP22 suppression remains unknown.     

Structural analysis of bovine mitochondrial tRNASer (AGY)

Mitochondrial serine tRNA lacking D stem (anticodon AGY) was purified from bovine cardiac muscle in the mg order. The nucleotide sequence was identical to that reported previously, with t6A adjacent to the 3' end of anticodon (Arcari, P. and Brownlee, G.G. (1980) Nucleic Acids Res. 8 5207-5212). The procedures for large scale preparation and some properties of this tRNASer are reported.     

Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm-replacement loop and a pseudo-tRNASer(UCN) gene.

A 2500-nucleotide pair (ntp) sequence of F-type mitochondrial (mt) DNA of the Pacific Rim mussel Mytilus californianus (class Bivalvia, phylum Mollusca) that contains two complete (ND2 and ND3) and two partial (COI and COIII) protein genes and nine tRNA genes is presented. Seven of the encoded tRNAs (Ala, Arg, His, Met(AUA), Pro, Ser(UCN), and Trp) have the potential to fold into the orthodox four-armed tRNA secondary structure, while two [tRNASer(AGN) and a second tRNASer(UCN)] will fold only into tRNAs with a dihydrouridine (DHU) arm-replacement loop. Comparison of these mt-tRNA gene sequences with previously published, corresponding M. edulis F-type mtDNA indicates that similarity between the four-armed tRNASer(UCN) genes is only 63.8% compared with an average of 92.1% (range 86.2-98. 5%) for the remaining eight tRNA genes. Northern blot analysis indicated that mature tRNAs encoded by the DHU arm-replacement loop-containing tRNASer(UCN), tRNASer(AGN), tRNAMet(AUA), tRNATrp, and tRNAPro genes occur in M. californianus mitochondria, strengthening the view that all of these genes are functional. However, Northern blot and 5' RACE (rapid amplification of cDNA ends) analyses indicated that the four-armed tRNASer(UCN) gene is transcribed into a stable RNA that includes the downstream COI sequence and is not processed into a mature tRNA. On the basis of these observations the M. californianus and M. edulis four-armed tRNASer(UCN) sequences are interpreted as pseudo-tRNASer(UCN) genes.     

Genetic analysis of structure and function in phage T4 tRNASer

We have determined the nucleotide sequences of 55 spontaneous mutations that inactivate a suppressor gene of phage T4 tRNASer. Most of the mutations caused substitutions or deletions of single nucleotides at 18 different positions in the tRNA. Two of three mutations that allowed the synthesis of mature tRNA had nucleotide substitutions at the junction of the dihydrouridine and anticodon stems, suggesting that this region of tRNASer is important for aminoacylation. The third mutation that synthesized tRNA had a nucleotide deletion in the anticodon loop, which presumably affected the translational capacity of the tRNA. We also sequenced 58 spontaneous reversion mutations derived from strains with the inactive suppressor genes. Some of these regenerated the initial tRNA sequence, while other generated a second-site mutation in the tRNA. These second-site mutations restored helical base-pairings to the tRNA that had been eliminated by the initial mutations. The new base-pairings involved G.C and A.U, and the A.C wobble pair at certain positions in the tRNA. This finding establishes the existence of A.C wobble pair in tRNA helices.     

Bovine mitochondrial tRNAPhe, tRNASer (AGY) and tRNASer (UCN): preparation using a new detection method and their properties in aminoacylation

Bovine mitochondrial tRNAPhe, tRNASer (AGY), and tRNASer (UCN) possessing unusual structures were purified using a new hybridization assay system and their properties in aminoacylation were examined. Bovine mitochondrial phenyl-alanyl- and seryl-tRNA synthetases could aminoacylate the same amino acid-specific tRNAs obtained not only from the mitochondria but also from other sources such as E. coli, Thermus thermophilus, bovine and yeast cytosols and archaebacteria, Sulfolobus acidocaldarius. On the contrary, none of both bacterial and cytosolic synthetases could aminoacylate the same amino acid specific tRNAs from the heterologous sources with some exceptions. We consider that the bovine mitochondrial aminoacyl-tRNA synthetases have considerably simple recognition mechanism toward the substrate tRNAs compared with the non-mitochondrial ones. This mechanism may be correlated with the occurrence of structural varieties of the mitochondrial tRNA species with unusual structures.     

Superposition of a tRNASer acceptor stem microhelix into the seryl-tRNA synthetase complex

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.     

Expression of bovine mitochondrial tRNASer GCU derivatives in Escherichia coli.

By replacing a stretch of five A-U base pairs in the acceptor stem with G-C pairs, mitochondrial tRNA-SerGCU lacking a D arm could be expressed in Escherichia coli cells in considerable amounts. The expressed tRNA with no modified nucleoside was serylated in vitro with the mitochondrial enzyme. The tRNASerGCU derivatives carrying identity elements for alanine tRNA and the related anticodons were expressed. However, this expression event did not affect cell growth, probably because the expression started from the late log phase, which suggests that these mitochondrial tRNA derivatives are not involved in E.coli gene expression systems. Although there are some restrictions in the secondary structure of tRNAs that can be expressed by this method, it could prove useful for preparing large amounts of heterologous tRNAs in vivo.     

Maize mitochondrial seryl-tRNA synthetase recognizes Escherichia coli tRNASer in vivo and in vitro

In our studies to analyze the structure/function relationships among cytoplasmic and organellar seryl-tRNA synthetases (SerRS), we have characterized a Zea mays cDNA (SerZMm) encoding a protein with significant similarity to prokaryotic SerRS enzymes. To demonstrate the functional identity of SerZMm, the gene sequence encoding the putative mature protein was cloned. This construct complemented in vivo a temperature-sensitive Escherichia coli serS mutant strain. The mature SerZMm protein overexpressed in Escherichia coli efficiently aminoacylated bacterial tRNA^Ser in vitro, while yeast tRNA was a poor substrate. These data identify SerZMm as an organellar maize seryl-tRNA synthetase, the first plant organellar SerRS to be cloned. The analysis of its N-terminal targeting signal suggests a mitochondrial function for the SerZMm protein in maize.     

Solution structure of a tRNA with a large variable region: yeast tRNASer

Different chemical reagents were used to study the tertiary structure of yeast tRNASer, a tRNA with a large variable region: ethylnitrosourea, which alkylates the phosphate groups; dimethylsulphate, which methylates N-7 of guanosine and N-3 of cytosine; and diethylpyrocarbonate, which modifies N-7 of adenine. The non-reactivity of N-3 of cytidine 47:1, 47:6, 47:7 and 47:8 and the reactivity of cytidine 47:3 confirms the existence of a variable stem of four base-pairs and a short variable loop of three residues. For the N-7 positions in purines, accessible residues are G1, G10, Gm18, G19, G30, I34, G35, A36, i6A37, G45, G47, G47:5, G47:9 and G73. The protection of N-7 atoms of residues G9, G15, A21, A22 and G47:9 reflects the tertiary folding. Strong phosphate protection was observed for P8 to P11, P20:1 to P22, P48 to P50 and for P59 and P60. A model was built on a PS300 graphic system on the basis of these data and its stereochemistry refined. While trying to keep most tertiary interactions, we adapted the tertiary folding of the known structures of tRNAAsp and tRNAPhe to the present sequence and solution data. The resulting model has the variable arm not far from the plane of the common L-shaped structure. A generalization of this model to other tRNAs with large variable regions is discussed.     

A study of the interaction between tRNASer and seryl-tRNA synthetase from bovine liver

Study by chemical modification of Ser, Arg, His residues and sulfhydryl groups on bovine seryl-tRNA synthetase showed that Ser residues appeared to be unnecessary for the recognition mechanism, but Arg and His residues were essential. It was considered that different sulfhydryl groups related with each recognition of tRNA and ATP. Poly-arginine inhibited the interaction between serine tRNA and SerRS. The CD spectra of a mixture of serine tRNA and poly-arginine indicated that higher-order structure of tRNA changed. Furthermore, the Km and Vmax values of bovine serine isoacceptor, yeast serine tRNA and E. coli serine tRNA for bovine SerRS examined and it was discussed the differences of those base sequences.     

C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes both tRNASer and tRNALeu

Leucyl-tRNA synthetase (LeuRS) is a multidomain enzyme that catalyzes Leu-tRNA^Leu formation and is classified into bacterial and archaeal/eukaryotic types with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNA^Leu through different modes of interaction. In the human pathogen Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNA^Ser (CatRNA^Ser(CAG)) in addition to its cognate CatRNA^Leu, leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNA^Ser, suggesting the significance of their highly divergent CTDs in tRNA^Ser recognition. The results of this study provided the first evidence of the indispensable function of the CTD of eukaryotic LeuRS in recognizing non-cognate CatRNA^Ser and cognate CatRNA^Leu. Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in the leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knock-out strain.     

Stronger affinity of reticulocyte release factor than natural suppressor tRNASer for the opal termination codon

Animal natural suppressor tRNA did not affect the release reaction of reticulocyte release factor (RF) at the same concentration of tRNA (both estimated as being present at a similar level of 3-5 X 10(-8) M in vivo); even at a 10-fold greater concentration the tRNA did not prevent the release reaction with RF. In order to confirm this result, the Ka values were determined. The Ka value between RF and UGA was 1.26 X 10(6) M-1 and that between the suppressor tRNA and UGA amounted to 8 X 10(3) M-1. This result showed that RF had a 150-fold stronger affinity than suppressor tRNA for the opal termination codon. Incorporation of phosphoserine into phosphoprotein via phosphoseryl-tRNA was inhibited by addition of RF to the reaction mixture. These results suggest that animal natural suppressor tRNA in the normal state does not perform its suppressor function, except in special cases where mRNA has the context structure near the opal termination codon (UGA).     

tRNASer(CGA) differentially regulates expression of wild-type and codon-modified papillomavirus L1 genes

Exogenous transfer RNAs (tRNAs) favor translation of bovine papillomavirus 1 wild-type (wt) L1 mRNA in in vitro translation systems (Zhou et al. 1999, J. Virol., 73, 4972–4982). We, therefore, investigated whether papillomavirus (PV) wt L1 protein expression could be enhanced in eukaryotic cells following exogenous tRNA supplementation. Both Chinese hamster ovary (CHO) and Cos1 cells, transfected with PV1 wt L1 genes, effectively transcribed the genes but did not translate them. However, L1 protein translation was demonstrated following co-transfection with the L1 gene and a gene expressing tRNA^Ser(CGA). Cell lines, stably transfected with a bovine papillomavirus 1 (BPV1) wt L1 expression construct, produced L1 protein after the transfection of the tRNA^Ser(CGA) gene, but not following the transfection with basal vectors, suggesting that tRNA^Ser(CGA) gene enhanced wt L1 translation as a result of endogenous tRNA alterations and phosphorylation of translation initiation factors elF4E and elF2α in the tRNA^Ser(CGA) transfected L1 cell lines. The tRNA^Ser(CGA) gene expression significantly reduced translation of L1 proteins expressed from codon-modified (HB) PV L1 genes utilizing mammalian preferred codons, but had variable effects on translation of green fluorescent proteins (GFPs) expressed from six serine GFP variants. The changes of tRNA pools appear to match the codon composition of PV wt and HB L1 genes and serine GFP variants to regulate translation of their mRNAs. These findings demonstrate for the first time in eukaryotic cells that translation of the target genes can be differentially influenced by the provision of a single tRNA expression construct.     

Processing of plant mitochondrial tRNAGly and tRNASer(GCU) is independent of RNA editing

The genes encoding pea and potato mitochondrial tRNA^Gly and pea mitochondrial tRNA^Ser(GCU) were analyzed with particular respect to their expression. Secondary-structure models deduced from the identical potato and pea tRNA^Gly gene sequences revealed A₇:C₆₆ mismatches in the seventh base pair at the base of the acceptor stems of both tRNAs. Sequence analyses of tRNA^Gly cDNA clones showed that these mispairings are not corrected by C₆₆ to U₆₆ conversions, as observed in plant mitochondrial tRNA^Phe. Likewise, a U₆:C₆₇ mismatch identified in the acceptor stem of the pea tRNA^Ser(GCU) is not altered by RNA editing to a mismatched U:U pair, which is created by RNA editing in Oenothera mitochondrial tRNA^Cys. In vitro processing reactions with the respective tRNA^Gly and tRNA^Ser(GCU) precursors show that such conversions are not necessary for 5′ and 3′ end maturation of these tRNAs. These results demonstrate that not all C:A (A:C) or U:C (C:U) mismatches in double-stranded regions of tRNAs are altered by RNA editing. An RNA editing event in plant mitochondrial tRNAs is thus not generally indicated by the presence of a mismatch but may depend on additional parameters.     

A precursor to a minor species of yeast tRNASer contains an intervening sequence.

Certain tRNAs in S. cerevisiae (tRNATyr and tRNAPhe) arise via precursor molecules which are mature at the 5' and 3' termini but contain intervening sequences adjacent to the anticodon (Knapp et al., 1978; O'Farrell et al., 1978). In addition to these molecules, precursors to several other tRNAs accumulate in a temperature-sensitive mutant (ts136) at the nonpermissive temperature. We have analyzed one of these species and shown that it is a precursor to a minor species of tRNASer. This precursor is also mature at both termini and contains an intervening sequence of 19 nucleotides adjacent to the hypermodified A residue 3' to the anticodon. The sequence can be arranged in a secondary structure in which the anticodon stem is extended by additional base-pairing, and contains the sites of excision and ligation within two looped regions. Support for this structure was provided by analysis of the products of limited digestion with RNAase T1. recently Piper (1978) reported the isolation of a minor species of tRNASer which decodes UCG. He found this species to be structurally heterogeneous and determined that the less abundant form corresponds to the tRNA which is altered in the recessive lethal SUP-RL1 amber suppressor. Our data now suggest that the more abundant form may be restricted to reading UCA in vivo; thus mutation of the minor species would result in complete loss of UCG-decoding ability and explain the recessive lethality of SUP-RL1. We have shown that the precursor which accumulates in ts136 corresponds exclusively to this minor tRNASerUCG species. Our results suggest that this may be the only gene for tRNASer in yeast which contains an intervening sequence.     

The unusual methanogenic seryl-tRNA synthetase recognizes tRNASer species from all three kingdoms of life.

The methanogenic archaea Methanococcus jannaschii and M. maripaludis contain an atypical seryl-tRNA synthetase (SerRS), which recognizes eukaryotic and bacterial tRNAsSer, in addition to the homologous tRNASer and tRNASec species. The relative flexibility in tRNA recognition displayed by methanogenic SerRSs, shown by aminoacylation and gel mobility shift assays, indicates the conservation of some serine determinants in all three domains. The complex of M. maripaludis SerRS with the homologues tRNASer was isolated by gel filtration chromatography. Complex formation strongly depends on the conformation of tRNA. Therefore, the renaturation conditions for in vitro transcribed tRNASer(GCU) isoacceptor were studied carefully. This tRNA, unlike many other tRNAs, is prone to dimerization, possibly due to several stretches of complementary oligonucleotides within its sequence. Dimerization is facilitated by increased tRNA concentration and can be diminished by fast renaturation in the presence of 5 mm magnesium chloride.     

Serine activation is the rate limiting step of tRNASer aminoacylation by yeast seryl tRNA synthetase.

Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.