The thermodynamic origin of the stability of a thermophilic ribozyme.
Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg(2+) to fold to their native structures that are very similar. The stability is measured as a function of Mg(2+) and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.
Fang
X W
XW
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637, USA.
Golden
B L
BL
Littrell
K
K
Shelton
V
V
Thiyagarajan
P
P
Pan
T
T
Sosnick
T R
TR
D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D004795:Enzyme Stability; D017665:Hydroxyl Radical; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D016337:RNA, Catalytic; D017665:Hydroxyl Radical; D014508:Urea; D008274:Magnesium