Nucleobase modification by an RNA enzyme.
Ribozymes can catalyze phosphoryl or nucleotidyl transfer onto ribose hydroxyls of RNA chains. We report a single ribozyme that performs both reactions, with a nucleobase serving as initial acceptor moiety. This unprecedented combined reaction was revealed while investigating potential contributions of ribose hydroxyls to catalysis by kinase ribozyme K28. For a 58nt, cis-acting form of K28, each nucleotide could be replaced with the corresponding 2΄F analog without loss of activity, indicating that no particular 2΄OH is specifically required. Reactivities of two-stranded K28 variants with oligodeoxynucleotide acceptor strands devoid of any 2΄OH moieties implicate modification on an internal guanosine N-2, rather than a ribose hydroxyl. Product mass suggests formation of a GDP(S) adduct along with a second thiophosphorylation, implying that the ribozyme catalyzes both phosphoryl and nucleotidyl transfers. This is further supported by transfer of radiolabels into product from both α and γ phosphates of donor molecules. Furthermore, periodate reactivity of the final product signifies acquisition of a ribose sugar with an intact 2΄-3΄ vicinal diol. Neither nucleobase modification nor nucleotidyl transfer has previously been reported for a kinase ribozyme, making this a first-in-class ribozyme. Base-modifying ribozymes may have played important roles in early RNA world evolution by enhancing nucleic acid functions.
Dept. of Biochemistry, University of Missouri, Columbia, MO, USA.|Bond Life Sciences Center, University of Missouri, Columbia, MO, USA
Raghav R
RR
Poudyal
Dept. of Biochemistry, University of Missouri, Columbia, MO, USA.|Bond Life Sciences Center, University of Missouri, Columbia, MO, USA
Phuong D M
PD
Nguyen
Bond Life Sciences Center, University of Missouri, Columbia, MO, USA|Department of Biological Engineering, University of Missouri, Columbia, MO, USA.
Melissa P
MP
Lokugamage
Bond Life Sciences Center, University of Missouri, Columbia, MO, USA|Department of Biological Engineering, University of Missouri, Columbia, MO, USA.
Mackenzie K
MK
Callaway
Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.
Jesse V
JV
Gavette
Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.
Ramanarayanan
R
Krishnamurthy
Dept. of Biochemistry, University of Missouri, Columbia, MO, USA.|Bond Life Sciences Center, University of Missouri, Columbia, MO, USA|Department of Biological Engineering, University of Missouri, Columbia, MO, USA.|Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO, USA
Donald H
DH
Burke
D001665:Binding Sites; D002384:Catalysis; D019143:Evolution, Molecular; D006151:Guanosine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D020871:RNA Stability; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D058615:Synthetic Biology; D013696:Temperature
D016337:RNA, Catalytic; D006151:Guanosine; D012313:RNA