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Titolo:
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
Autore:
Chen, X; Gutell, RR; Lambowitz, AM;
Indirizzi:
Univ Texas, Inst Cellular & Mol Biol, Dept Chem & Biochem, Sch Biol Sci, Austin, TX 78712 USA Univ Texas Austin TX USA 78712 iochem, Sch Biol Sci, Austin, TX 78712 USA Univ Texas, Mol Genet Sect, Sch Biol Sci, Austin, TX 78712 USA Univ TexasAustin TX USA 78712 t Sect, Sch Biol Sci, Austin, TX 78712 USA Univ Texas, Microbiol & Integrat Biol Sect, Sch Biol Sci, Austin, TX 78712USA Univ Texas Austin TX USA 78712 ol Sect, Sch Biol Sci, Austin, TX 78712USA
Titolo Testata:
JOURNAL OF MOLECULAR BIOLOGY
fascicolo: 2, volume: 301, anno: 2000,
pagine: 265 - 283
SICI:
0022-2836(20000811)301:2<265:FOTSIS>2.0.ZU;2-V
Fonte:
ISI
Lingua:
ENG
Soggetto:
TRANSFER-RNA-SYNTHETASE; COMPARATIVE SEQUENCE-ANALYSIS; TERTIARY STRUCTURE; TETRAHYMENA RIBOZYME; ESCHERICHIA-COLI; CATALYTIC CORE; TD INTRON; RIBOSOMAL-RNA; MISMATCHES; PHAGE-T4;
Keywords:
aminoacyl-tRNA synthetase; group I intron; ribozyme; RNA splicing; RNA structure;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
55
Recensione:
Indirizzi per estratti:
Indirizzo: Lambowitz, AM Univ Texas, Inst Cellular & Mol Biol, Dept Chem & Biochem, Sch Biol Sci, Austin, TX 78712 USA Univ Texas Austin TX USA 78712 ol Sci, Austin, TX 78712 USA
Citazione:
X. Chen et al., "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", J MOL BIOL, 301(2), 2000, pp. 265-283

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 ateither P4 bp-1 or P6 bp-1, for mutations at P6 bp1 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 theanalysis 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)-EDTAanalysis 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. (C) 2000 Academic Press.

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Documento generato il 06/04/20 alle ore 05:40:11