The Neurospora mitochondrial tyrosyl-tRNA synthetase is sufficient for group I intron splicing in vitro and uses the carboxy-terminal tRNA-binding domain along with other regions.
Neurospora mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by nuclear gene cyt-18, functions in splicing of group I introns in mitochondria. Here, we overproduced functional cyt-18 protein in Escherichia coli and purified it to near homogeneity. The purified protein has splicing and tyrRS activities similar to those of cyt-18 protein isolated from mitochondria and is by itself sufficient to splice the mitochondrial large rRNA intron in vitro. Structure-function relationships in the cyt-18 protein were analyzed by in vitro mutagenesis. We confirmed that a small amino-terminal domain not found in bacterial tyrRSs is required for splicing activity, but not tyrRS activity. Two linker insertion mutations, which disrupt the predicted ATP-binding site, completely inhibit tyrRS activity but leave substantial splicing activity. Finally, deletions or linker insertion mutations in the putative carboxy-terminal tRNA-binding domain inhibit both tyrRS and splicing activities, although some have differential effects on the two activities. Our results show that the normal catalytic activity of the cyt-18 protein is not required for splicing and are consistent with the hypothesis that the protein functions by binding to the precursor RNA and facilitating formation of the correct RNA structure. Regions required for splicing are distributed throughout the cyt-18 protein and overlap, but are not identical to, regions required for tyrRS activity. The finding that the putative carboxy-terminal tRNA-binding domain is required for both tyrRS and splicing activities suggests that the mechanism for binding the intron has similarities to the mechanism for binding tRNA(Tyr).
Kittle
J D
JD
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Mohr
G
G
Gianelos
J A
JA
Wang
H
H
Lambowitz
A M
AM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D005800:Genes, Fungal; D007438:Introns; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D015183:Restriction Mapping; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D012331:RNA, Fungal; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D014448:Tyrosine-tRNA Ligase