A TYROSYL-TRANSFER-RNA SYNTHETASE PROTEIN INDUCES TERTIARY FOLDING OFTHE GROUP-I INTRON CATALYTIC CORE

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 pr otein) functions in splicing group I introns. We have used chemical-st ructure mapping and footprinting to investigate the interaction of the CYT-18 protein with the N. crassa mitochondrial large subunit ribosom al RNA (mt LSU) and ND1 introns, which are not detectably self-splicin g 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 la rgely unfolded, even at high Mg2+ concentrations. The binding of CYT-1 8 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 b inding results in the protection of regions of the phosphodiester back bone expected for tertiary folding of the catalytic core, as well as a dditional protections that may reflect proximity of the protein. In bo th 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-1 8 as a separate RNA molecule, but additional sites are found in the ot her 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 stru cture and splicing activity. Considered together with previous studies , our 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 do main, which may contain additional binding sites for the protein. A th ree-dimensional model structure of the CYT-18-binding site in group I introns indicates that CYT-18 interacts with the surface of the cataly tic 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. (C) 1996 Academic Press Limited