Assembly of an exceptionally stable RNA tertiary interface in a group I ribozyme

Biochemistry. 1999 Mar 9;38(10):2982-90. doi: 10.1021/bi982113p.

Abstract

Group I intron RNAs contain a core of highly conserved helices flanked by peripheral domains that stabilize the core structure. In the Tetrahymena group I ribozyme, the P4, P5, and P6 helices of the core pack tightly against a three-helix subdomain called P5abc. Chemical footprinting and the crystal structure of the Tetrahymena intron P4-P6 domain revealed that tertiary interactions between these two parts of the domain create an extensive solvent-inaccessible interface. We have examined the formation and stability of this tertiary interface by providing the P5abc segment in trans to a Tetrahymena ribozyme construct that lacks P5abc (EDeltaP5abc). Equilibrium gel shift experiments show that the affinity of the P5abc and EDeltaP5abc RNAs is exceptionally strong, with a Kd of approximately 100 pM at 10 mM MgCl2 (at 37 degrees C). Chemical and enzymatic footprinting shows that the RNAs are substantially folded prior to assembly of the complex. Solvent accessibility mapping reveals that, in the absence of P5abc, the intron RNA maintains a nativelike fold but its active-site helices are not tightly packed. Upon binding of P5abc, the catalytic core becomes more tightly packed through indirect effects of the tertiary interface formation. This two-component system facilitates quantitative examination of individual tertiary contacts that stabilize the folded intron.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Macromolecular Substances
  • Molecular Sequence Data
  • Nucleic Acid Conformation*
  • Nucleic Acid Denaturation
  • RNA, Catalytic / chemistry*
  • RNA, Protozoan / chemistry*
  • Spectrophotometry, Ultraviolet
  • Tetrahymena thermophila / enzymology
  • Thermodynamics

Substances

  • Macromolecular Substances
  • RNA, Catalytic
  • RNA, Protozoan