Assembly of an Exceptionally Stable RNA Tertiary Interface in a Group I Ribozyme

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...

Full description

Saved in:
Bibliographic Details
Published in:Biochemistry (Easton) 1999-03, Vol.38 (10), p.2982-2990
Main Authors: Doherty, Elizabeth A, Herschlag, Daniel, Doudna, Jennifer A
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Catalysis
Crystal structure
Gels
Interfaces (materials)
Macromolecular Substances
Molecular Sequence Data
Molecular structure
Nucleic Acid Conformation
Nucleic Acid Denaturation
RNA, Catalytic - chemistry
RNA, Protozoan - chemistry
Solvents
Spectrophotometry, Ultraviolet
Tetrahymena
Tetrahymena thermophila - enzymology
Thermodynamics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 (EΔP5abc). Equilibrium gel shift experiments show that the affinity of the P5abc and EΔP5abc RNAs is exceptionally strong, with a K d of ∼100 pM at 10 mM MgCl2 (at 37 °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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi982113p