Nucleotide sequences of yeast genes for tRNA(2), tRNA(2) and tRNA(1): homology blocks occur in the vicinity of different tRNA genes

Three members of a collection of pBR322-yeast DNA recombinant plasmids containing yeast tRNA genes have been analyzed and sequenced. Each plasmid carries a single tRNA gene: pY44, tRNA^Ser₂; pY41, tRNA^Arg₂; pY7, tRNA^Val₁. All three genes are intronless and terminate in a cluster of Ts in the non-coding strand. The sequence information here and previously determined sequences allow an extensive comparison of the regions flanking several yeast tRNA genes. This analysis has revealed novel features in tRNA gene arrangement. Blocks of homology in the flanking regions were found between the tRNA genes of an isoacceptor family but, more interestingly, also between genes coding for tRNAs of different amino-acid specificities. Particularly, three examples are discussed in which sequence elements in the neighborhood of different tRNA genes have been conserved to a high degree and over long distances.     

The first human genes for tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU) and more tRNA(Val) pseudogenes: expression and pre-tRNA maturation in HeLa cell-free extracts.

A functional tRNA(Val) gene, which codes for the major tRNA(ValIAC) isoacceptor species, and three new tRNA(Val) pseudogenes have been isolated from human genomic DNA. Two tRNA(Val) pseudogenes and a tRNA(Val) variant gene were found to be associated with tRNA genes encoding tRNA(ArgICG), tRNA(GlyUCC), and tRNA(ThrIGU), respectively, on distinct DNA fragments. All tRNA genes, including the pseudogenes, are actively transcribed in HeLa nuclear extract. Pre-tRNAs of tRNA(Val), tRNA(Arg), tRNA(Thr), and tRNA(Gly) genes are correctly processed to mature-sized tRNAs, whereas the three tRNA(Val) pseudogenes yield stable pre-tRNAs in vitro. These findings reveal that, together with the three known pseudogenes, half of the members of the human tRNA(Val) gene family are pseudogenes, all of which are active in homologous nuclear extracts in vitro and presumably also in vivo.     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.     

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.     

Isolation of novel tRNA(Ala) mutants by library selection in a tRNA(Ala) knockout strain

The relationship between tRNA structure and function has been widely investigated by site-directed mutagenesis. This method has been a very useful tool to reveal the critical bases in tRNAs that are important for recognition and aminoacylation, but has been limited by the large number of possible base combinations in tRNA molecules. We have devised a new method that uses tRNA knockout cells for selection of functional tRNAs from a mutant tRNA gene library to overcome this limitation. To explore the mechanism of tRNA(Ala) recognition, the bases of the acceptor-stem region were randomized and active mutants were selected in a tRNA(Ala) knockout strain. Mutants of tRNA(Ala) having diverse sequence combinations in the acceptor-stem region and a broad range of functional activity to support knockout cell growth were isolated. The mutant tRNAs selected by the method included molecules containing novel base substitutions as well as extensively altered base combinations that would not be readily generated by rationally designed site-directed mutagenesis. Our results emphasize the importance of the acceptor stem as a structural unit in which some nucleotides may carry more weight than others, but in summation every nucleotide contributes to the interaction with the enzyme.     

The Catalytic Domain of Topological Knot tRNA Methyltransferase (TrmH) Discriminates between Substrate tRNA and Nonsubstrate tRNA via an Induced-fit Process

A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2′-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.