A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core.
The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used an Escherichia coli genetic assay based on the phage T4 td intron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long-range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in the td intron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.
Myers
C A
CA
Department of Molecular Genetics, Ohio State University 43210-1292, USA.
Wallweber
G J
GJ
Rennard
R
R
Kemel
Y
Y
Caprara
M G
MG
Mohr
G
G
Lambowitz
A M
AM
D017122:Bacteriophage T4 / Q000737:chemistry; D001483:Base Sequence; D002874:Chromosome Mapping; D004926:Escherichia coli; D007438:Introns; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D009690:Nucleic Acid Conformation; D010641:Phenotype; D012326:RNA Splicing; D012367:RNA, Viral / Q000737:chemistry*; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry*
D012367:RNA, Viral; D014448:Tyrosine-tRNA Ligase