The proposed research involves biochemical, genetic, and structural studies of how cellular proteins assist in the folding and splicing of group I introns. Group I introns are catalytic RNAs that self-splice in vitro but require proteins for efficient splicing in vivo, to help the RNA fold into the catalytically active RNA structure. In Neurospora crassa, a nuclear-encoded mitochondrial tyrosyl-tRNA synthetase (CYT-18) and a DEAD-box protein (CYT-19) are required for splicing three mitochondrial group I introns in vivo, and this splicing can be recapitulated in vitro with purified components. In the proposed research, this model system will be used to investigate how DEAD-box proteins are recruited to and mediate RNA conformational changes on a natural RNP substrate. Since DEAD-box proteins are involved in virtually all cellular processes involving RNA, results of this study will be widely applicable to understanding the mode of action of other DEAD-box proteins.
Specific aims are: (1) To investigate the interaction of CYT-19 with CYT-18 and intron RNA. (2) To identify the regions of CYT-19 responsible for targeting CYT-18-dependent group I introns and the regions of CYT-18 interacting with CYT-19. (3) To determine the structure of CYT-19 by X-ray crystallography. ? ?
