Efficient protein-facilitated splicing of the yeast mitochondrial bI5 intron.
The splicing factor CBP2 is required to excise the yeast mitochondrial group I intron bI5 in vivo and at low magnesium ion concentrations in vitro. CBP2 binding is strengthened 20-fold by increasing Mg2+ concentrations from 5 to 40 mM, implying the protein binds, in part, to the same structure as that stabilized by the cation. The same transition is also observed as a cooperative increase in the rate of self-processing between 5 and 40 mM Mg2+, providing strong evidence for an RNA folding transition promoted by either Mg2+ or CBP2. The first step of splicing, guanosine addition at the 5' splice site, is rate limiting for exon ligation. At low (5 mM) magnesium ion, reaction (measured as kcat/Km or kcat) is accelerated 3 orders of magnitude by saturating CBP2. At near-saturating Mg2+ (40 mM), acceleration is 8- and 30-fold, for kcat and kcat/Km, respectively, so high magnesium ion concentrations fail to compensate completely for protein facilitation. Thus, self-splicing proceeds via two additional transitions as compared with reaction of the bI5-CBP2 complex, only the first of which is efficiently promoted by the cation. Guanosine 5'-monophosphate binds (Kd approximately 0.3 mM) with the same affinity to bI5 and the bI5-protein complex, supporting independent binding of the nucleophile and CBP2. Substitution of a phosphorothioate at the 5' splice site and pH profiles provide evidence that kcat is limited by chemistry at low pH and by a conformational step at high pH. Because binding by either Mg2+ or CBP2 increases the rate of chemistry more than the rate of the conformational step, in the physiological pH range (7-7.6) the protein-facilitated reaction is limited by a conformation step while self-splicing reaction is limited by chemistry. We conclude that CBP2 makes manifold contributions to bI5 splicing: binding compensates for at least two structural defects and accelerates the rate of the chemistry.
Weeks
K M
KM
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215, USA.
Cech
T R
TR
D001483:Base Sequence; D005656:Fungal Proteins / Q000378:metabolism*; D006860:Hydrogen Bonding; D007438:Introns; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics*; D012326:RNA Splicing; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins / Q000737:chemistry; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
C071249:CBP2 protein, S cerevisiae; D005656:Fungal Proteins; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D008274:Magnesium