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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 coil 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 TM.[Key Words: RNA splicing; group I intron; RNA catalysis; tyrosyl-tRNA synthetase; aminoacyl-tRNA synthetase; mitochondrial RNA processing] Received January 24, 1991; revised version accepted March 27, 1991.The Neurospora mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by nuclear gene cyt-18, functions in splicing the large rRNA intron and other group I introns in Neurospora mitochondria (Akins and Lambowitz 1987;Majumder et al. 1989). As discussed elsewhere, the most likely hypothesis is that proteinassisted splicing of group I introns remains RNA catalyzed and proteins function primarily to facilitate correct folding of the intron RNA Lambowitz and Perlman 1990). Because the cyt-18 protein is an aminoacyl-tRNA synthetase, we suggested that it might bind to the intron RNA via recognition of a sequence or structure that resembles the aminoacylation substrate tRNA TM (Akins and Lambowitz 1987).IOn leave from Lehrstuhl fiir Allgemeine Botanik, Ruhr University of Bochum, Germany. 2Corresponding author.To investigate in more detail the involvement of tyrRS in splicing, we carried out biochemical analysis of the tyrRS protein isolated from mitochondria (Majumder et al. 1989). Using antibodies against trpE-cyt-18 fusion proteins, we identified the cyt-18 gene product as a basic protein having a relative molecular mass of -70 kD. We then show...