In vivo analysis of the relationships between the splicing and homing activities of a group I intron-encoded I-ScaI/bi2-maturase of Saccharomyces capensis produced in the yeast cytoplasm.
The I-ScaI/bi2-maturase of Saccharomyces capensis acts as a specific homing endonuclease promoting intron homing, and as a maturase promoting intron splicing. Using the universal code equivalent of the mitochondrial gene encoding the I-ScaI/bi2-maturase, a number of truncated forms of the synthetic gene were constructed, shortened on either side, as were several mutated alleles of the protein. The shortest translation product that fully retains both activities in vivo corresponds to 228 codons of the C-terminal region of the bi2 intron-encoded protein, whereas proteins resulting from more extensive deletions either at the N-terminus or at the C-terminus (up to 73 and four residues, respectively) were able to complement wholly the lack of endogenous maturase, but all lost the endonuclease activity. Similarly, all introduced mutations completely abolished the I-ScaI activity while some mutant proteins retained substantial splicing function. Immunodetection experiments demonstrated that different cytoplasmically translated forms of the I-ScaI/bi2-maturase protein were imported into mitochondria and correctly processed. They appeared to be tightly associated with mitochondrial membranes. Homology modelling of the I-ScaI/bi2-maturase protein allowed us to relate both enzymatic activities to elements of enzyme structure.
Szczepanek
Tomasz
T
Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, France.
Gora
Monika
M
Monteilhet
Claude
C
Wysocka
Monika
M
Lazowska
Jaga
J
Golik
Pawel
P
D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000201:enzymology*; D004272:DNA, Mitochondrial / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D007150:Immunohistochemistry; D007438:Introns; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012326:RNA Splicing; D012440:Saccharomyces / Q000201:enzymology*
D004272:DNA, Mitochondrial; D004722:Endoribonucleases; C414590:Bi2 maturase; D003850:Deoxyribonuclease I