A trans acting ribozyme that phosphorylates exogenous RNA.
The structural complexity required for substrate recognition within an active site constrains the evolution of novel catalytic functions. To evaluate those constraints within populations of incipient ribozymes, we performed a selection for kinase ribozymes under conditions that allowed competition for phosphorylation at nine candidate sites. Two candidate sites are the hydroxyl groups on a "quasi-diffusible" chloramphenicol (Cam) moiety tethered to the evolving library through an inert, flexible linker. A subtractive step was included to allow only seven ribose 2' hydroxyls to compete with the two Cam hydroxyls for phosphorylation. After the library was incubated with gamma-thio-ATP (ATPgammaS), active species were recovered from a polyacrylamide gel containing [(N-acryloylamino)phenyl] mercury (APM) and amplified for further cycles of selection. Activity assays on selected isolates and truncated derivatives identified the essential secondary structure of the dominant RNA motif. Phosphorylation was independent of the Cam moiety, indicating ribose 2' phosphorylation. The dominant motif was separated into catalytic "ribozyme" and "substrate" strands. Partial alkaline digestion of the substrate strand before and after phosphorylation identified the precise modification site as the first purine (R) within the required sequence 5'-RAAAANCG-3'. The reaction shows approximately 10-fold preference for ATPgammaS over ATP and is independent of pH over a wide range (5.5-8.9), consistent with a dissociative reaction mechanism that is rate-limited by formation of a metaphosphate transition state. Divalent metal ions are required, with a slight preference of Mn(2+) > Mg(2+) > Ca(2+). Lack of reactivity in [Co(NH(3))(6)](3+) indicates a requirement for inner sphere contact with the metal ion, either for structural stabilization, catalysis, or both.
Saran
Dayal
D
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, USA.
Nickens
David G
DG
Burke
Donald H
DH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent / Q000737:chemistry; D004252:DNA Mutational Analysis; D019020:Directed Molecular Evolution; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
C506620:2PTmin3.2 ribozyme; D002413:Cations, Divalent; D016337:RNA, Catalytic; C022571:adenosine 5'-O-(3-thiotriphosphate); D012313:RNA; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor)