Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymatic properties pertinent to splicing.
The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in the splicing of group I introns. Here, bacterially expressed CYT-18 protein, purified by a new procedure involving polyethyleneimine precipitation to remove tightly bound nucleic acids, was used to characterize properties pertinent to RNA splicing. Analytical ultracentrifugation and other methods showed that the CYT-18 protein is an asymmetric homodimer. The measured frictional ratio, f/fo = 1.55, corresponds to an axial ratio of 10 for a prolate ellipsoid or 12 for an oblate ellipsoid. Like bacterial TyrRSs, the CYT-18 protein exhibits half-sites reactivity, each homodimer having one active site for tyrosyl adenylation and RNA splicing. The splicing activity of CYT-18 was unaffected by aminoacylation substrates at concentrations used in aminoacylation reactions, whereas the TyrRS activity was inhibited by physiological concentrations of the splicing cofactor GTP, as well as CTP or UTP, or by low concentrations of a group I intron RNA. Kinetic measurements suggest that the binding of CYT-18 to a group I intron substrate is a two-step process, with an initial biomolecular step that is close to diffusion limited (3.24 +/- 0.03 x 10(7) M-1s-1) followed by a slower conformational change (0.54 +/- 0.07 s-1). After CYT-18 binding, splicing occurs at a rate of 0.0025 s-1, within 6-fold of the rate of self-splicing of the Tetrahymena large rRNA intron in vitro. The Kd for the complex between the CYT-18 protein and a group I intron substrate, calculated from koff/kon, was < 0.3 pM, substantially lower than determined by presumed equilibrium measurements [Guo, Q., & Lambowitz, A. M. (1992) Genes Dev. 6, 1357-1372]. As a result of this tight binding, the CYT-18 protein functions stoichiometrically in in vitro splicing reactions due to its extremely slow dissociation from the excised intron RNA. The very tight binding of the CYT-18 protein to the intron RNA raises the possibility that specific mechanisms exist for dissociating the protein from the excised intron in vivo.
Saldanha
R J
RJ
Department of Molecular Genetics, Ohio State University, Columbia 43210-1292.
Patel
S S
SS
Surendran
R
R
Lee
J C
JC
Lambowitz
A M
AM
D003001:Cloning, Molecular; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007438:Introns; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing; D000077278:RNA, Mitochondrial; D012343:RNA, Transfer / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D000077278:RNA, Mitochondrial; D011994:Recombinant Proteins; D014443:Tyrosine; D012313:RNA; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D014448:Tyrosine-tRNA Ligase