Efficient Protein-Facilitated Splicing of the Yeast Mitochondrial bI5 Intron

Biochemistry. 1995 Jun 13;34(23):7728-38. doi: 10.1021/bi00023a020.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Fungal Proteins / metabolism*
  • Hydrogen Bonding
  • Introns
  • Kinetics
  • Magnesium / metabolism
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • RNA / genetics*
  • RNA Splicing*
  • RNA, Mitochondrial
  • RNA-Binding Proteins / chemistry
  • Recombinant Proteins
  • Ribonucleoproteins*
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins*
  • Thermodynamics

Substances

  • CBP2 protein, S cerevisiae
  • Fungal Proteins
  • RNA, Mitochondrial
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Ribonucleoproteins
  • Saccharomyces cerevisiae Proteins
  • RNA
  • Magnesium