Back to Search
Start Over
Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme.
- Source :
-
RNA (New York, N.Y.) [RNA] 2006 Dec; Vol. 12 (12), pp. 2118-25. Date of Electronic Publication: 2006 Oct 26. - Publication Year :
- 2006
-
Abstract
- 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.
- Subjects :
- Adenosine Triphosphate analogs & derivatives
Adenosine Triphosphate metabolism
Base Sequence
Kinetics
Magnesium chemistry
Magnesium metabolism
Molecular Sequence Data
Phosphorylation
Polynucleotide 5'-Hydroxyl-Kinase chemistry
Polynucleotide 5'-Hydroxyl-Kinase metabolism
Temperature
Thermodynamics
RNA, Catalytic chemistry
RNA, Catalytic metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1355-8382
- Volume :
- 12
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- RNA (New York, N.Y.)
- Publication Type :
- Academic Journal
- Accession number :
- 17068208
- Full Text :
- https://doi.org/10.1261/rna.173506