Kinetic and thermodynamic framework for assembly of the six-component bI3 group I intron ribonucleoprotein catalyst.
The yeast mitochondrial bI3 group I intron RNA splices in vitro as a six-component ribonucleoprotein complex with the bI3 maturase and Mrs1 proteins. We report a comprehensive framework for assembly of the catalytically active bI3 ribonucleoprotein. (1) In the absence of Mg(2+), two Mrs1 dimers bind independently to the bI3 RNA. The ratio of dissociation to association rate constants, k(off)/k(on), is approximately equal to the observed equilibrium K(1/2) of 0.12 nM. (2) At magnesium ion concentrations optimal for splicing (20 mM), two Mrs1 dimers bind with strong cooperativity to the bI3 RNA. k(off)/k(on) is 15-fold lower than the observed K(1/2) of 11 nM, which reflects formation of an obligate intermediate involving one Mrs1 dimer and the RNA in cooperative assembly of the Mrs1-RNA complex. (3) The bI3 maturase monomer binds to the bI3 RNA at almost the diffusion-controlled limit and dissociates with a half-life of 1 h. k(off)/k(on) is approximately equal to the equilibrium K(D) of 2.8 pM. The bI3 maturase thus represents a rare example of a group I intron protein cofactor whose binding is adequately characterized by a one-step mechanism under conditions that promote splicing. (4) Maturase and Mrs1 proteins each bind the bI3 RNA tightly, but with only modest coupling (approximately 1 kcal/mol), suggesting that the proteins interact at independent RNA binding sites. Maturase binding functions to slow dissociation of Mrs1; whereas prior Mrs1 binding increases the bI3 maturase k(on) right to the diffusion limit. (5) At effective concentrations plausibly present in yeast mitochondria, a predominant assembly pathway emerges involving rapid, tight binding by the bI3 maturase, followed by slower, cooperative assembly of two Mrs1 dimers. In the absence of other factors, disassembly of all protein subunits will occur in a single apparent step, governed by dissociation of the bI3 maturase.
Bassi
Gurminder S
GS
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Weeks
Kevin M
KM
D002384:Catalysis; D002499:Centrifugation, Density Gradient; D019281:Dimerization; D007438:Introns; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics*; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
C078505:MRS1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins