Kinetic and secondary structure analysis of Naegleria andersoni GIR1, a group I ribozyme whose putative biological function is site-specific hydrolysis.
NanGIR1 is a catalytic element inserted in the P6 loop of a group I intron (NanGIR2) in the small subunit rRNA precursor of the protist Naegleria andersoni [Einvik, C., Decatur, W. A., Embley, T. M., Vogt, V. M., and Johansen, S. (1997) RNA 3, 710-720]. It catalyzes site-specific hydrolysis at an internal processing site (IPS) after a G residue that immediately follows the P9 stem-loop. Functional and structural analyses were initiated to compare NanGIR1 to group I introns that carry out self-splicing. Chemical modification and site-directed mutagenesis studies showed that NanGIR1 shares many structural elements with other group I introns, but also contains a pseudoknot (P15), which is important for catalytic activity. Deletion analysis revealed the boundaries of the minimum self-cleaving unit (178 nucleotides). The rate of self-cleavage was measured as a function of mono- and divalent ion concentration, temperature, and pH. The reaction at the IPS yields 5'-phosphate and 3'-hydroxyl termini, requires Mg2+or Mn2+ ions, and is first-order in [OH-] between pH 5.0 and 8.5. The latter results suggest that the nucleophile in the reaction is hydroxide or possibly a Mg2+-coordinated hydroxide. With a second-order rate constant of 1 x 10(5) min-1 M-1, the self-cleavage reaction of NanGIR1 is 2 orders of magnitude faster than a similar site-specific hydrolysis reaction of the circular form of the Tetrahymena group I intron.
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Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215, USA.
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D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007438:Introns / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009253:Naegleria / Q000737:chemistry* / Q000201:enzymology; D009690:Nucleic Acid Conformation; D011188:Potassium / Q000494:pharmacology; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics
D012322:RNA Precursors; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D008345:Manganese; D008274:Magnesium; D011188:Potassium