Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase with group I intron RNAs

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, or CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. To investigate how CYT-18 stabilizes the active RNA structure, I used an Escherichia coli genetic assay with the phage T4 td intron to systematically test the ability of the CYT-18 protein to compensate for structural defects in the P7 region of the group I intron core. P7 is a long-range base-pairing interaction of the P3-P9 domain that forms the binding site for the splicing co-factor guanosine. My results show that CYT-18 can suppress numerous mutations that impair the self-splicing of the td intron, including mutations that disrupt base-pairing within the P7 region. 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 some selected mutant introns showed that the P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. I used directed hydroxyl radical cleavage assays to show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNATyr cocrystal structure.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Ecology, Evolution, and Behavior

Grantor

The University of Texas at Austin

Year dc:date.issued

2002

Author dc:creator

• Myers, Christopher Allan

Advisor dc:contributor.advisor

• Lambowitz, Alan

Statement dc:rights

• Copyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.

Language dc:language.iso

eng

Identifier

b57170137

OAI identifier oai:identifier

oai:repositories.lib.utexas.edu:2152/807