Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase with group I introns
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The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, or CYT-18 protein, functions in splicing by binding specifically to the group I intron catalytic core. To investigate how the CYT-18 promotes group I intron splicing, I used an Escherichia coli genetic assay based on the phage T4 td intron to test the ability of CYT-18 to suppress mutations that cause structural defects around its binding site in the P4-P6 domain of the group I intron catalytic core. My results show that most mutations at either P4 bp-1 or P6 bp-1 inhibit self-splicing, but can be suppressed by CYT-18. CYT-18 can compensate efficiently for mutations that disrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-1 that disrupt the base-triple interaction with J3/4-3, and for nucleotide substitutions at either position that are predicted to be suboptimal for base stacking. Mutations at the junction of the P4-P6 stacked helices lead to grossly impaired tertiary-structure formation centered in the P4-P6 domain. CYT-18-suppressiable mutants bind the protein with increased Kd up to 79-fold while the koff values are within twofold. My results indicate that the P4-P6 junction is a linchpin region in group I intron and that CTY-18 binding induces the formation of the correct structure in this region, leading to folding of the group I intron catalytic core. Using a similar genetic system, I also show that the tertiary interactions of P5- L9 and P8-L2 are important for group I intron splicing. CYT-18 C-terminal domain is required for suppression of the mutations that disrupt these tertiary interactions. Together with previous RNA footprinting and modeling data, my results suggest that P8 in group I intron RNA might contact with regions in the C-terminal domain of the CYT-18 protein and that the interactions between CYT-18 and both P4-P6 and P3-P9 domains help stabilize the tertiary interactions in group I intron and promote the splicing of group I introns.