TY - JOUR
T1 - Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength
AU - Brown, Trevor S.
AU - Chadalavada, Durga M.
AU - Bevilacqua, Philip C.
N1 - Funding Information:
This work was supported by National Institutes of Health grant GM58709, a fellowship from the Alfred P. Sloan Foundation (to P.C.B.), a Camille Dreyfus Teacher-Scholar Award (to P.C.B.), an NRSA Individual Fellowship from the NIH (to T.S.B.) and an Alfred P. Sloan Scholarship (to T.S.B.).
PY - 2004/8/13
Y1 - 2004/8/13
N2 - 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/U27Δ 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/U27Δ in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs≈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 Mg 2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence.
AB - 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/U27Δ 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/U27Δ in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs≈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 Mg 2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence.
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U2 - 10.1016/j.jmb.2004.05.071
DO - 10.1016/j.jmb.2004.05.071
M3 - Article
C2 - 15288780
AN - SCOPUS:4344584210
SN - 0022-2836
VL - 341
SP - 695
EP - 712
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -
