A tyrosyl-tRNA synthetase protein induces tertiary folding of the group I intron catalytic core.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns. We have used chemical-structure mapping and footprinting to investigate the interaction of the CYT-18 protein with the N. crassa mitochondrial large subunit ribosomal RNA (mt LSU) and ND1 introns, which are not detectably self-splicing in vitro. Our results show that both these non-self-splicing introns form most of the short range pairings of the conserved group I intron secondary structure in the absence of CYT-18, but otherwise remain largely unfolded, even at high Mg2+ concentrations. The binding of CYT-18 promotes the formation of the extended helical domains P6a-P6-P4-P5 (P4-P6 domain) and P8-P3-P7-P9 (P3-P9 domain) and their interaction to form the catalytic core. In iodine-footprinting experiments, CYT-18 binding results in the protection of regions of the phosphodiester backbone expected for tertiary folding of the catalytic core, as well as additional protections that may reflect proximity of the protein. In both introns, most of the putative CYT-18 protection sites are in the P4-P6 domain, the region of the SU intron previously shown to bind CYT-18 as a separate RNA molecule, but additional sites are found in the other major helical domain in P8 and P9 in both introns and in L9 and P7.1/P7.1a in the mt LSU intron. Protease digestion of the CYT-18/intron RNA complexes results in the loss of CYT-18-induced RNA tertiary structure and splicing activity. Considered together with previous studies, or results suggest that CYT-18 binds initially to the P4-P6 region of group I introns to form a scaffold for the assembly of the P3-P9 domain, which may contain additional binding sites for the protein. A three-dimensional model structure of the CYT-18 binding site in group I introns indicates that CYT-18 interacts with the surface of the catalytic core on the side opposite the active-site cleft and may primarily recognize a specific three-dimensional geometry of the phosphodiester backbone of group I introns.