Viva Origino Vol.41 No.2

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Yoh-ichi Watanabe1* and Shigeo Yoshinari1,2
1Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
2Present address: Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA.
*Corresponding author: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan,
Fax: +81-3-5841-3444, e-mail:


   DNA replication is a fundamental phenomenon to maintain and transfer of the genetic information in the living organisms. Elucidation of the molecular mechanism of DNA replication has been one of the main subjects since molecular biology started. Most of the research results in the field were obtained from Escherichia coli and its phages in an early stage. Then, research was expanded to the eukaryotic cells, including yeast and mammalian cells. It is now well recognized that living organisms are divided into three domains. Archaea, the third domain, different from Bacteria and Eukarya, was joined to the field of DNA replication after two other domains and the research has been active since late 1990’s to present. Comparative studies in the three domains of life provide much useful information to understand the evolution of DNA replication machinery. In this mini-review article, we will discuss mainly the molecular evolution of DNA polymerase in the living organisms.

(Keywords) DNA replication, Archaea, DNA polymerase, molecular evolution


   In Archaea, almost all introns in pre-tRNA, pre-rRNA, and pre-mRNA are spliced through two common steps by protein enzymes: cleavage of the precursor with splicing endonuclease, and ligation of the exons with RNA ligase. We found the first examples of archaeal pre-mRNA splicing and cleavage of the pre-mRNA with a novel subclass of archaeal splicing endonuclease. We further solved the novel tertiary structure of the splicing endonuclease, and revealed that the lineage-specific insertion of amino acid residues in the endonuclease expands the recognition of the substrate precursor RNA. We also discuss the possible involvement of tRNA splicing and its machinery in the origin of the tRNA molecule.

intron, RNA splicing, Archaea, splicing endonuclease, RNA ligase, bulge-helix-bulge motif


    In bacteria and eukaryotes, some types of RNA splicing occur by precursor RNAs themselves, or with the aid of trans-factors including RNA-protein complexes [1]. However, in Archaea, with a few exceptions of group II introns found in Euryarchaeota [2], almost all of the introns in pre-tRNA [3], pre-rRNA [4], and pre-mRNA [5] are thought to be spliced through two steps catalyzed by protein-based enzymes: cleavage at exon-intron boundaries in precursor RNA with splicing endonuclease [6], and ligation of the exon fragments with RNA ligase [7].
   The splicing endonuclease recognizes a specific structure in the substrate precursor RNA bulge-helix-bulge (BHB) motif, composed of a 4-basepair central helix flanked by two 3-base bulge loops at the 3’ sides together with basepairs, and cleaves the specific sites in the “bulge” region [8, Fig. 1A]. The recognition of the substrate with the enzyme is independent of the other part of the substrate, including the majority of the exon region [9]. Archaeal splicing endonucleases are classified into three subclasses based on subunit structures: homo-tetrameric enzymes, homo-dimeric enzymes, and hetero-tetrameric (alpha2beta2) enzymes [6]. Archaeal splicing endonuclease shares a common ancestor with eukaryotic tRNA splicing endonuclease [10].
   The recently identified archaeal RNA ligase [11] shares a common ancestor with a bacterial RNA repair enzyme [12] and a human tRNA ligase [13]. Archaeal RNA ligase joins the 3’ end of the 5’ exon with a 2’,3’-cyclic phosphate residue and the 5’ end of the 3’ exon without a phosphate residue, and uses the phosphate residue at the 3’ end of the 5’ exon as the linkage between the exons [14].

Fig 1. BHB motifs predicted in archaeal cbf5 pre-mRNAs [15]. A, a conventional BHB motif in Aeropyrum pernix cbf5 pre-mRNA. B, a relaxed BHB motif in Sulfolobus tokodaii cbf5 pre-mRNA. Rectangles in blue represent the central 4-bp helices. Triangles in green represent the 3-base bulge loops. Note that in S. tokodaii pre-mRNA, the C residue at the 3’ side of the 3’ bulge (indicated in red) cannot form a Watson-Crick basepair with the A residue (also in red) at the 5’ side of the central helix. Arrows represent cleavage sites with the splicing endonuclease confirmed in vitro [5,18]. Upper case, exon regions; lower case, intron regions.

Discovery of archaeal pre-mRNA splicing, and identification of structural elements in archaeal splicing endonuclease for expansion of substrate RNA recognition

    In 2002, we identified the first examples of introns in archaeal protein coding genes [15]. Further analysis revealed that pre-mRNA fragments were cleaved at the exon-intron boundaries with heteromeric crencarcheal splicing endonuclease [5]. Notably, some of the exon-intron boundaries in orthologous genes in related species may not form strict BHB motifs, including mismatches in the helices and/or bulge loops with fewer or more than 3 bases (Fig. 1B) [5,15,16]. Conventional euryarchaeal homo-tetrameric and homo-dimeric splicing endonucleases cannot cleave such relaxed forms of the BHB motif [6]. To reveal the structural elements that expand the substrate recognition in the crenarchaeal heteromeric enzyme, we determined the crystal structures of two crenarchaeal enzymes, and found that they take a hetero-tetrameric (alpha2beta2) subunit structure [17,18]. Moreover, we showed that the specific insertion of amino acid sequences in the crenarchaeal enzymes, rather than the subunit structure of the crenarchaeal enzymes, is important for substrate recognition [17,18]. Transplantation of the insertion sequences into a euryarchaeal homo-dimeric enzyme expanded substrate recognition [19,20, Yoshinari, S. and Watanabe, Y. unpublished results]. A specific lysine residue in the inserted sequence is important for the substrate recognition/catalysis of the enzyme [18,19,20]. Although the nanoarchaeal enzyme with the expanded substrate recognition does not have the insertion at the corresponding position [21], a novel insertion at the alternative position may have a similar role [18].

tRNA splicing and the origin of tRNA

   It has been proposed that tRNA was born by a fusion of two half RNA molecules which included a single stem-loop flanked by single strand regions at both the 5’ and 3’ sides [22]. Interestingly, some of the eukaryotic pre-tRNAs including introns in the anticodon loops have autocatalytic hydrolysis at or near the position of exon-intron boundaries [23], suggesting that a pre-tRNA molecule with an intron may be related to the prototypical tRNA. It would be interesting to investigate the autocatalytic hydrolysis of archaeal pre-tRNAs. Furthermore, many bacteria have homologs of archaeal and eukaryotic (t)RNA ligase, although bacteria do not have tRNA splicing driven by splicing endonucleases and RNA ligases [7], suggesting that this type of RNA ligase may exist in the last common ancestor of the three domains of life, and may have played a role in forming a prototype tRNA from two tRNA half molecules.


   Our studies were partly supported by the metabolic protein group of the National Project on Protein Structural and Functional Analyses, and Grants-in-Aid of the Ministry of Science, Sports and Culture, Japan to YW and SY.


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