Functionally distinct nucleic acid binding sites for a group I intron encoded RNA maturase/DNA homing endonuclease.
A large number of group I introns encode a family of homologous proteins that either promote intron splicing (maturases) or are site-specific DNA endonucleases that function in intron mobility (a process called "homing"). Genetic studies have shown that some of these proteins have both activities, yet how a single protein carries out both functions remains obscure. The similarity between respective DNA-binding sites and the RNA structure near the 5' and 3' splice sites has fueled speculation that such proteins may use analogous interactions to perform both functions. The Aspergillus nidulans mitochondrial COB group I intron encodes a bi-functional protein, I-AniI, that has both RNA maturase and site-specific DNA endonuclease activities in vitro. Here, we show that I-AniI shows distinctive features of the endonuclease family to which it belongs, including highly specific, tight binding and sequential DNA strand cleavage. Competition experiments demonstrate that I-AniI binds the COB intron RNA even in saturating concentrations of its DNA target site substrate, suggesting that the protein has a separate binding site for RNA. In addition, we provide evidence that two different DNA-binding site mutants of I-AniI have little effect on the protein's RNA maturation activity. Since RNA splicing is likely a secondary adaptation of the protein, these observations support a model in which homing endonucleases may have developed maturase function by utilizing a hitherto "non-functional" protein surface.
Chatterjee
Piyali
P
Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4960, USA.
Brady
Kristina L
KL
Solem
Amanda
A
Ho
Yugong
Y
Caprara
Mark G
MG
D019943:Amino Acid Substitution; D001233:Aspergillus nidulans / Q000201:enzymology*; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D007438:Introns / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D004271:DNA, Fungal; D010446:Peptide Fragments; D012331:RNA, Fungal; D012194:RNA-Directed DNA Polymerase; C454879:maturase I-AniI; D004720:Endonucleases