@article {387,
	title = {Function of tyrosyl-tRNA synthetase in splicing group I introns: an induced-fit model for binding to the P4-P6 domain based on analysis of mutations at the junction of the P4-P6 stacked helices.},
	journal = {J Mol Biol},
	volume = {301},
	number = {2},
	year = {2000},
	month = {2000 Aug 11},
	pages = {265-83},
	abstract = {We used an Escherichia coli genetic assay based on the phage T4 td intron to test the ability of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) to suppress mutations that cause structural defects around its binding site in the P4-P6 domain of the group I intron catalytic core. We analyzed all possible combinations of nucleotides at either P4 bp-1 or P6 bp-1, which together form the junction of the P4-P6 stacked helices, and looked for synergistic effects in double mutants. Most mutations at either position 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, based on the analysis of DNA four-way junctions. However, CYT-18 has difficulty suppressing combinations of mutations at P4 bp-1 and P6 bp-1 that simultaneously disrupt base-pairing and base stacking. Thermal denaturation and Fe(II)-EDTA analysis showed that 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-suppressible mutants bind the protein with K(d) values up to 79-fold higher than that for the wild-type intron, but in all cases tested, the k(off) value for the complex remains within twofold of the wild-type value, suggesting that the binding site can be formed properly and that the increased K(d) value reflects primarily an increased k(on) value for the binding of CYT-18 to the misfolded intron. Our results indicate that the P4/P6 junction is a linchpin region, where even small nucleotide substitutions grossly disrupt the catalytically-active group I intron tertiary structure, and that CYT-18 binding induces the formation of the correct structure in this region, leading to folding of the group I intron catalytic core.},
	keywords = {Bacteriophage T4, Catalytic Domain, Edetic Acid, Escherichia coli, Introns, Models, Biological, Mutation, Neurospora crassa, Nucleic Acid Conformation, Protein Denaturation, Protein Structure, Secondary, Protein Structure, Tertiary, RNA Splicing, Suppression, Genetic, Tyrosine-tRNA Ligase},
	issn = {0022-2836},
	doi = {10.1006/jmbi.2000.3963},
	author = {Chen, X and Gutell, R R and Lambowitz, A M}
}
