Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme.
The Kin.46 ribozyme catalyzes transfer of the gamma (thio)phosphoryl group of ATP (or ATPgammaS) to the ribozyme's 5' hydroxyl. Single-turnover catalytic activities of topologically rearranged versions of Kin.46 were studied to gain insight into its overall tertiary architecture. The distal ends of stems P3 and P4 were tethered through a single-stranded connection domain that altered the interhelical connectivity. The shortest linkers interfered with catalysis, while seven or more nucleotides (nt) in the linker allowed near-normal catalytic rates, suggesting that a distance of roughly 25-35 A optimally separates the termini of these helices. Activity was maximal when the tether contained 15 nt, at which point the k(cat) (0.016 min(-1)) and Km (1.2 mM) values were identical to those of a nontethered control. The presence of the tether alters Mg(2+) dependence, in that Mg2+ binding appears to be more cooperative in the tethered ribozyme (Hill coefficient 1.4-1.8 versus 0.8 for the nontethered ribozyme). Binding affinity for the ATPgammaS substrate increases at elevated concentrations of Mg2+, particularly for the tethered ribozyme. The tethered ribozyme displays significantly enhanced thermal stability, with a maximum initial velocity (0.126 min(-1)) at 60 degrees C, whereas the nontethered ribozyme has a lower maximum initial velocity (0.051 min(-1)) at 50 degrees C. The tether also significantly reduces the apparent entropy of activation. Both of these effects can be understood in terms of stabilization of the ribozyme in a conformation that is on-path with respect to catalysis, and in terms of facilitating formation of the allosteric activation helix P4.
Cho
Bongrae
B
Department of Applied Chemistry, Division of Applied Science, Cheongju University, Cheongju 360-764, Korea.
Burke
Donald H
DH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D016337:RNA, Catalytic; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D008274:Magnesium