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Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength.

Abstract：

:The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA that resides in the HDV genome and regulates its replication. The native fold of the ribozyme is complex, having two pseudoknots. Earlier work implicated four non-native pairings in slowing pseudoknot formation: Alt 1, Alt 2, Alt 3, and Alt P1. The goal of the present work was design of a kinetically simplified and maximally reactive construct for in vitro mechanistic and structural studies. The initial approach chosen was site-directed mutagenesis in which known alternative pairings were destabilized while leaving the catalytic core intact. Based on prior studies, the G11C/U27Delta double mutant was prepared. However, biphasic kinetics and antisense oligonucleotide response trends opposite those of the well-studied G11C mutant were observed suggesting that new alternative pairings with multiple registers, termed Alt X and Alt Y, had been created. Enzymatic structure mapping of oligonucleotide models supported this notion. This led to a model wherein Alt 2 and the phylogenetically conserved Alt 3 act as "folding guides", facilitating folding of the major population of the RNA molecules by hindering formation of the Alt X and Alt Y registers. Attempts to eliminate the strongest of the Alt X pairings by rational design of a quadruple mutant only resulted in more complex kinetic behavior. In an effort to simultaneously destabilize multiple alternative pairings, studies were carried out on G11C/U27Delta in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs approximately 60 min(-1)) kinetics to be realized. To account for this, a model is developed wherein Na+, which destabilizes secondary and tertiary structures in the presence of Mg2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Brown TS,Chadalavada DM,Bevilacqua PC

doi

10.1016/j.jmb.2004.05.071

keywords:

subject

Has Abstract

pub_date

2004-08-13 00:00:00

pages

695-712

issue

eissn

0022-2836

issn

1089-8638

pii

S0022283604006527

journal_volume

341

pub_type

杂志文章

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Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength.