Two Latent Homing Endonucleases Encoded in the rRNA Introns of the Hyperthermophilic Archaeon Aeropyrum pernix K1
Norimichi NOMURA*, Yoshihiko SAKO, Yayoi MORINAGA, and Aritsune UCHIDA
Laboratory of Marine Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-01, Japan
Regardless of the phylogenetic arguments for early or late origin of introns, their mobility might have played and/or play a pivotal role as driving force of genome evolution. Our ultimate goal is to elucidate the mechanism for propagation and tolerance of introns in hyperthermophilic archaeal systems. This study focuses on the functional aspect of the intron-encoded proteins of the hyperthermophilic archaeon Aerophrum pernix K1.
In a separate study, we show that three introns (Iα, Iβ, and Iγ) reside within the rRNA operon of A. perinix K1. Two of them, Iα and Iγ, contain long ORFs consist of 221 and 137 amino acids, respectively, and the following circumstantial evidences suggest that they are expressed in vivo: I) only one long reading frame is possible for each intron, ii) putative Shine-Dalgarno sequences occur 10-15 upstream of the start codons, iii) post-splicing, free intron RNAs which can function as m RNAs are stably accumulated in the cell, although their relative quantity fluctuate in dependence on the physiological conditions. Comparative sequence analyses against databases revealed no genes or proteins with any overall similarity. However, a search made with each dodecapeptide sequence3, 127YVKGFVDAEGSV138 in Iα ORF(I-Ape I) and 9YVAGIIDAEASL20 in IγORF, revealed a similar motif (LAGLI-DADG) characteristic of proteins encoded by group I introns and inteins.
In this context, we have overexpressed the IαORF in E. coli and purified the recombinant I-Ape I protein to homogeneity by heat treatment and chromatography. I-Ape I recognize the double stranded DNA whose sequence is 5'
GGCTGAAAC*TTAAAGG3' and cleaves as indicated by the asterisk, generating four nucleotide 3'-overhangs. The enzyme reaches peak activity at temperatures ranging from 85 to 90℃ and requires Mg.2+ Considering that the target sequence corresponds to that lies at intron-insertion site of an intronless 16S rRNA gene of Aeropyrum pernix OH2, our result raised the possibility that I-Ape I might also Aeropyrum pernix OH2, our result raised the possibility that I-Ape I might also promote intron mobility, as was demonstrated for the Saccharomyces cerevisiae mitochondrial aI4α intronic ORF. Now, we are also developing the overexpression system of IγORF in E. coli to confirm the intron-homing endonucleolytic activity.
