Format

Send to:

Choose Destination
See comment in PubMed Commons below
RNA Biol. 2013 Mar;10(3):425-35. doi: 10.4161/rna.23609. Epub 2013 Jan 16.

Intermolecular domain docking in the hairpin ribozyme: metal dependence, binding kinetics and catalysis.

Author information

  • 1Department of Biochemistry and Molecular Biology; Michigan State University; East Lansing, MI USA.

Abstract

The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH 3) 6 (3+). RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a "double conformational capture" model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.

KEYWORDS:

RNA catalysis; RNA-cation interactions; circular dichroism; hairpin ribozyme; ribozyme kinetics; surface plasmon resonance

PMID:
23324606
[PubMed - indexed for MEDLINE]
PMCID:
PMC3672286
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Landes Bioscience Icon for PubMed Central
    Loading ...
    Write to the Help Desk