Protein-dependent transition states for ribonucleoprotein assembly

J Mol Biol. 2001 Jun 22;309(5):1087-100. doi: 10.1006/jmbi.2001.4714.


Native folding and splicing by the Saccharomyces cerevisiae mitochondrial bI5 group I intron RNA is facilitated by both the S. cerevisiae CBP2 and Neurospora crassa CYT-18 protein cofactors. Both protein-bI5 RNA complexes splice at similar rates, suggesting that the RNA active site structure is similar in both ribonucleoproteins. In contrast, the two proteins assemble with the bI5 RNA by distinct mechanisms and bind opposing, but partially overlapping, sides of the group I intron catalytic core. Assembly with CBP2 is limited by a slow, unimolecular RNA folding step characterized by a negligible activation enthalpy. We show that assembly with CYT-18 shows four distinctive features. (1) CYT-18 binds stably to the bI5 RNA at the diffusion controlled limit, but assembly to a catalytically active RNA structure is still limited by RNA folding, as visualized directly using time-resolved footprinting. (2) This mechanism of rapid stable protein binding followed by subsequent assembly steps has a distinctive kinetic signature: the apparent ratio of k(off) to k(on), determined in a partitioning experiment, differs from the equilibrium K(d) by a large factor. (3) Assembly with CYT-18 is characterized by a large activation enthalpy, consistent with a rate limiting conformational rearrangement. (4) Because assembly from the kinetically trapped state is faster at elevated temperature, we can identify conditions where CYT-18 accelerates (catalyzes) bI5 RNA folding relative to assembly with CBP2.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Allosteric Site
  • Base Sequence
  • Catalysis
  • Catalytic Domain
  • Fungal Proteins / metabolism*
  • Hydroxyl Radical / metabolism
  • Introns / genetics
  • Iodine / metabolism
  • Kinetics
  • Models, Molecular
  • Neurospora crassa
  • Nucleic Acid Conformation*
  • Protein Binding
  • RNA / chemistry
  • RNA / genetics
  • RNA / metabolism
  • RNA Splicing / genetics*
  • RNA Stability
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / genetics
  • RNA, Catalytic / metabolism
  • RNA, Mitochondrial
  • RNA-Binding Proteins / metabolism*
  • Ribonucleoproteins / chemistry
  • Ribonucleoproteins / genetics*
  • Ribonucleoproteins / metabolism*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins*


  • CBP2 protein, S cerevisiae
  • Fungal Proteins
  • RNA, Catalytic
  • RNA, Mitochondrial
  • RNA-Binding Proteins
  • Ribonucleoproteins
  • Saccharomyces cerevisiae Proteins
  • Hydroxyl Radical
  • RNA
  • Iodine