Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks

RNA. 1996 Jul;2(7):718-32.

Abstract

The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Cross-Linking Reagents
  • Magnesium / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • RNA, Catalytic / chemistry*
  • RNA, Catalytic / metabolism
  • RNA, Protozoan / chemistry
  • RNA, Protozoan / metabolism
  • Sodium / metabolism
  • Temperature
  • Tetrahymena / genetics*
  • Ultraviolet Rays

Substances

  • Cross-Linking Reagents
  • RNA, Catalytic
  • RNA, Protozoan
  • Sodium
  • Magnesium