Mechanism for tRNA splicing in eukaryotes

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In Eubacteria, tRNA introns are self-splicing group I and Group II introns. In Eukarya and Archaea, tRNA splicing is catalysed by the sequential action of specialised enzymes. The tRNA splicing reaction in yeast occurs in three steps:

  1. Substrate recognition and intron excision

    The first step does not require ATP. It involves phosphodiester bond cleavage, and is an atypical nuclease reaction. It is catalysed by an endonuclease.

    Pre-tRNA is cleaved at its two splice sites by an endonuclease. The products of the endonuclease reaction are the two tRNA half-molecules with 2',3'-cyclic phosphate (2',3'- P) and 5'-OH ends, and the linear intron with 5'-OH and 3'-phosphate (3'-P) ends.

    Although details of pre-tRNA substrate recognition differ between organisms, the endonucleases from Eukarya and Archaea are closely phylogenetically related. The precise cleavage at the two splice sites is explained by a ?ruler mechanism? in which a fixed distance to the splice sites is measured from a certain position in the mature domains of the tRNAs. The two splice sites are cleaved independently and the two "half" tRNA pieces stay together via H-bonding.

  2. Joining of both tRNA half-molecules: A baroque reaction

    The second step requires ATP and involves bond formation; it is a ligation reaction, and the responsible enzymes activity is described as an RNA ligase. When ATP is added, the second reaction occurs. Both of the unusual ends generated by the endonuclease must be altered.

    In a first step, the 2',3' cyclic phosphate (2',3'cyclic PO4) is opened to generate a 2'-phosphate terminus. This requires a cyclic phosphodiesterase (CPDase). The product has a 2'-phosphate (2'-PO4) group and a 3'OH group.

    In a second step, the 5'-OH group generated by the endonuclease must be phosphorylated to give a 5'-phosphate. This requires a kinase which phosphorylates the 5'-OH of the 3'-exon using the - PO4 of GTP (requires ATP).

    In a third step, the tRNA half molecules fold into a tRNA-like structure that now has a 3'-OH, 5'-P break. It is sealed by a ligase.

    An Adenylate synthetase (ASTase) adenylates the 3' half-molecules and activate the ligase using ATP. tRNA ligase is adenylated at an active site lysine, and then the AMP is transferred to the 5'-PO4 of the substrate. Formation of the 5'-3'-phosphodiester bond proceeds and AMP is released.

  3. Removal of the extra 2'-phosphate

    The spliced molecule is now uninterrupted, with a 5'-3' phosphate linkage at the site of splicing, but it is also has a 2'-phosphate group that must be removed by a phosphatase to complete the splicing reaction.

    In a final step, a nicotinamide adenine dinucleotide (NAD)-dependent 2' phosphotransferase (2'-Ptase) removes the 2' phosphate by transfer to NAD, generating a mature tRNA with a standard 3'-5' linkage at the splice junction. The structure of the transfer product is ADP-ribose 1'-2'-cyclic phosphate. The nicotinamide moiety is displaced, apparently supplying the energy for cyclisation.

Note: Two different mechanisms have been observed in eukaryotes for the joining steps of tRNA splicing:
  1. In the yeast S. cerevisiae, tRNA ligase joins the half-molecules to generate a splice junction bearing 2'-phosphate, and 2'-phostransferase transfers the 2'-phosphate to NAD to form ADP ribose 1'-2' cyclic phosphate. This ligase/phosphotransferase pathway is universal.
  2. The vertebrates also appear to have a second and completely different ligase, which directly joins the 5'-OH and the cyclic phosphate ends of the half-molecules to generate a junction with a normal 3'-5' phosphodiester bond but no extra 2'-phosphate.

Last modified: Tue Feb 10 11:21:12 CET 2004