Journal of Molecular Biology
Regular articleUnique recognition style of tRNALeu by Haloferax volcaniiLeucyl-tRNA synthetase1
Introduction
The fidelity of translation is maintained by the correct interaction between a given codon and its corresponding anticodon, and by a specific aminoacylation of a tRNA with a cognate amino acid. The former is guaranteed by Watson-Crick or wobble base-pairings, while the latter depends on the specific recognition of both amino acid and tRNA by aminoacyl-tRNA synthetase. Each of 20 aminoacyl-tRNA synthetases must recognize only its cognate tRNA isoacceptors specific for an amino acid while discriminating other remaining non-cognate tRNA molecules with apparently indistinguishable tertiary structures. Considerable progress has been made in understanding these mechanisms; the recognition elements that are crucial for the specific recognition of cognate tRNAs and those of which transplantation into other tRNAs can change their amino acid specificities. In most cases, these elements are located at the acceptor stem, the discriminator base, the anticodon, and sometimes the variable pocket of a tRNA Schulman 1991, Shimizu et al 1992, Giege et al 1998. They are often conserved among species Edwards and Schimmel 1987, Hou and Schimmel 1989, Weygand-Durasevic et al 1993, Aphasizhev et al 1996, while some divergence has been reported Trezeguet et al 1991, Nazarenko et al 1992, Nameki et al 1992, Nameki et al 1995, Nameki et al 1997, Moor et al 1995.
The recognition manner of tRNA is thought to have undergone evolution coupled with changes in the structure and the number of tRNA molecules. Although most tRNAs have a relatively uniform structure due to many constraints in the translational processes, they can be classified into two classes according to the length of the variable arm. Class I (type 1) tRNAs have a short variable arm comprising four or five nucleotides, while class II (type 2) tRNAs have a long variable arm composed of more than ten nucleotides. Based on this classification, a clear line can be drawn between prokarya and eukarya/archea. Eubacteria and organelles from lower eukaryotes have three class II tRNAs (tRNASer, tRNALeu and tRNATyr), while eukaryote cytoplasm and archaebacteria have only two class II tRNAs (tRNASer and tRNALeu). As an unusual example, metazoan mitochondria have no such tRNA. Because of this phylogenetic divergence in tRNA structural classification, the class II tRNA system appears to be a good model for studying the evolution of tRNA recognition Hartlein and Cusack 1995, Lenhard et al 1999.
Our previous studies have shown that the recognition manner of class II tRNA differs substantially between Escherichia coli and the yeast Saccharomyces cerevisiae, as if it reflects the disparity in the member of class II tRNAs between them. E. coli SerRS and LeuRS adopt a unique recognition style Himeno et al 1990, Asahara et al 1991, Asahara et al 1993, Asahara et al 1994. SerRS recognizes the long variable arm of tRNASer but not the discriminator base (G73) or the anticodon. LeuRS recognizes the discriminator base (A73) but not the anticodon nucleotides, despite the absolute conservation of the second base (A35) within the tRNALeu isoacceptors. Among the three aminoacyl-tRNA synthetases for class II tRNAs, only TyrRS recognizes the discriminator base and the anticodon like many other synthetases for class I tRNAs. In addition to these positive recognition elements, SerRS and LeuRS depend on a unique discrimination style in which they distinguish cognate and non-cognate class II tRNAs on the basis of the shape of a tRNA molecule rather than its specific nucleotide sequence. The conversion of amino acid acceptor specificity from either tRNALeu or tRNATyr to a serine acceptor, or from either tRNASer or tRNATyr to a leucine acceptor, requires changes of a set of elements that are involved in the tertiary folding; R15-Y48, R59, the location of the invariant G18G19 sequence in the D-loop, and the variable number of the unpaired nucleotides at the base of the variable arm Himeno et al 1990, Asahara et al 1993, Asahara et al 1994.
On the contrary, yeast class II tRNAs adopt a discrimination manner substantially different from E. coli. Yeast SerRS principally recognizes the variable arm (Himeno et al., 1997), and LeuRS recognizes some nucleotides in the anticodon loop as well as the discriminator base (Soma et al., 1996). Insertion of only one nucleotide into the variable loop is sufficient to confer serine acceptor activity on tRNALeu, and tRNASer can successfully acquire leucine acceptor activity simply by base substitutions at the discriminator base and the anticodon arm. These amino acid acceptor identity introductions do not require any change of tertiary element.
The discrimination manner is apparently stringent or exclusive in E. coli, while it is less exclusive in yeast. This has been demonstrated clearly in a cross-species aminoacylation study (Soma & Himeno, 1998). SerRS and LeuRS from E. coli cannot aminoacylate any yeast class II tRNAs, while yeast aminoacyl-tRNA synthetases can efficiently aminoacylate E. coli class II tRNAs (Soma & Himeno, 1998). This unilaterality of aminoacylation specificity between E. coli and yeast seems to reflect the differences in the number and the composition of their class II tRNAs, and thus the evolution of the recognition manner is thought to have been greatly affected by the number of non-cognate tRNAs. From these considerations, as well as from the unilateral aminoacylation specificity between eubacteria and animal mitochondria (Kumazawa et al., 1991), we proposed that the higher the number of similar-shaped tRNAs, the more exclusive the recognition/discrimination system becomes. It needs to be clarified whether this tendency is generally observed in the class II tRNA system from other sources.
The archaea system is expected to provide a clue for the understanding of the evolution of the class II tRNA recognition system. Archaea occupy a phylogenetically interesting location in the unrooted Woesian tree that emerged from ribosomal RNA sequence comparisons (Woese, 1987), and have apparent hybrid features in that the system of gene expression is similar to that of eukaryotes while the metabolic system is similar to that of prokaryotes (Brown & Doolittle, 1997). The aminoacylation system of archaea has recently become a subject of considerable interest; the identification of a methanogenic archaeal LysRS as a class I synthetase (Ibba et al., 1997), the utilization of other NTPs by AspRS in a hyperthermophilic archaea (Fujiwara et al., 1996), the formation of tRNA-dependent Asn-tRNAAsn in a halophile (Curnow et al., 1996), and the paraphyletic origin of the SerRS gene in the archaea kingdom Kim et al 1998, Lenhard et al 1999. However, little is known about the tRNA identity determination in archea as yet.
In this study, we focused on the recognition system of leucyl-tRNA synthetase from an extreme halophilic archaebacterium, Haloferax volcanii, belonging to the group euryarchaeota. It is intriguing to know whether the recognition strategy of class II tRNA in archaea is prokaryotic or eukaryotic. The composition of class II tRNA (tRNASer and tRNALeu) in H. volcanii raises the possibility that SerRS and LeuRS have a base-specific recognition manner, as in yeast. Alternatively, H. volcanii may adopt a tertiary structure-dependent manner as in E. coli, considering that the compositions of tertiary structural elements differ between tRNASer and tRNALeu.
To clarify this problem, we attempted to identify the determinants of tRNALeu from H. volcanii using unmodified tRNA transcripts constructed by T7 RNA polymerase in vitro(Sampson & Uhlenbeck, 1988). This study revealed a unique recognition manner of H. volcanii LeuRS, which is partly similar to that of E. coli, but is distinct from that of yeast.
Section snippets
The unmodified tRNALeu from H. volcanii
Usually the tRNA transcript free of modification serves as a good substrate for cognate aminoacyl-tRNA synthetase, with some exceptions such as E. coli tRNALys, tRNAIle and tRNAGluTamura et al 1992, Muramatsu et al 1992, Sylvers et al 1993. The H. volcanii tRNALeu used in this study has several archaebacteria-specific modified nucleotides Gupta 1984, Gupta 1986, such as archaeosine in the D-loop (Watanabe et al., 1997) and 1-methylinosine in the TΨC-loop (Grosjean et al., 1995). The unmodified
Discussion
The present study revealed that the discriminator base (A73) and the long variable arm, especially the specific loop sequence A47CG47D and U47H at the base of this helix, are significant for recognition by LeuRS from a halophilic archaea, H. volcanii. The elements involved in recognition of class II tRNAs from E. coli, S. cerevisiae and H. volcanii are summarized in Table 4.
The discriminator base is the only position that is conserved as the identity determinant of tRNALeu among species. On the
Preparation of template DNAs and in vitro transcripts
Synthetic DNA oligomers carrying the tRNA gene under the phage T7 promoter sequence were ligated into pUC19 and transformed into E. coli strain JM109 Sampson and Uhlenbeck 1988, Himeno et al 1989. The template DNA sequences were confirmed by dideoxy sequencing (Messing, 1983). Each template DNA of the discriminator base-substituted mutant was prepared from a plasmid carrying the wild-type tRNA sequence and two synthetic primers, by mutation via the polymerase chain reaction (Himeno et al., 1990)
Acknowledgements
We would like to thank Drs T. Yokogawa, T. Suzuki, N. Nameki, N. Okada, M. Ishigami, T. Hasegawa, H. Ihara, K. Watanabe, S. Ejiri, C. Ushida and A. Muto for their kind advice and helpful discussions. We also thank the Gene Research Center of Hirosaki University for the use of the facility. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan.
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Edited by D. E. Draper
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Present addresses: Teruyuki Sakamoto, The Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 Japan; Miho Maeda, Hokuriku National Agricultural Experiment Station, 1-2-1, Inada, Joetsu, Niigata, 943-01 Japan.