A trans acting ribozyme that phosphorylates exogenous RNA.

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Catalysis

Background:

• Enzyme active site complexity limits the evolution of new catalytic functions.
• Ribozymes, RNA molecules with catalytic activity, offer a model for studying the evolution of novel functions.

Purpose of the Study:

• To investigate the evolutionary constraints on developing new kinase ribozyme functions.
• To identify RNA motifs capable of catalyzing phosphorylation reactions.

Main Methods:

• In vitro selection was used to isolate kinase ribozymes from an evolving RNA library.
• Competition assays were performed against candidate phosphorylation sites, including chloramphenicol and ribose hydroxyls.
• Active ribozymes were recovered using polyacrylamide gel electrophoresis and mercury affinity chromatography.
• Structural analysis involved separating ribozyme and substrate strands and partial alkaline digestion to pinpoint the modification site.

Main Results:

• Selection yielded dominant RNA motifs capable of phosphorylating RNA substrates.
• Phosphorylation occurred specifically at the first purine (R) in the sequence 5'-RAAAANCG-3', independent of the chloramphenicol moiety.
• The reaction demonstrated a preference for ATPgammaS over ATP and was pH-independent, suggesting a dissociative mechanism.
• Divalent metal ions, particularly Mn(2+), were required, indicating a need for inner sphere metal ion contact.

Conclusions:

• The study identified specific RNA motifs that function as kinase ribozymes, phosphorylating RNA at a defined purine site.
• Evolutionary constraints on active site structure influence the development of novel catalytic activities.
• The characterized ribozyme reaction mechanism provides insights into RNA-based catalysis and the evolution of enzymes.