Nucleic Acids Research Advance Access published online on November 29, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm1055
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Nucleic Acid Enzymes |
Mechanisms of covalent self-assembly of the Azoarcus ribozyme from four fragment oligonucleotides
Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA
*To whom correspondence should be addressed. Tel: +1 503 725 8769; Fax: +1 503 725 9525; Email: niles{at}pdx.edu
Received September 19, 2007. Revised October 16, 2007. Accepted November 7, 2007.
RNA oligomers of length 40–60 nt can self-assemble into covalent versions of the Azoarcus group I intron ribozyme. This process requires a series of recombination reactions in which the internal guide sequence of a nascent catalytic complex makes specific interactions with a complement triplet, CAU, in the oligomers. However, if the CAU were mutated, promiscuous self-assembly may be possible, lessening the dependence on a particular set of oligomer sequences. Here, we assayed whether oligomers containing mutations in the CAU triplet could still self-construct Azoarcus ribozymes. The mutations CAC, CAG, CUU and GAU all inhibited self-assembly to some degree, but did not block it completely in 100 mM MgCl2. Oligomers containing the CAC mutation retained the most self-assembly activity, while those containing GAU retained the least, indicating that mutations more 5' in this triplet are the most deleterious. Self-assembly systems containing additional mutant locations were progressively less functional. Analyses of properly self-assembled ribozymes revealed that, of two recombination mechanisms possible for self-assembly, termed ‘tF2’ and ‘R2F2’, the simpler one-step ‘tF2’ mechanism is utilized when mutations exist. These data suggest that self-assembling systems are more facile than previously believed, and have relevance to the origin of complex ribozymes during the RNA World.
Present address: Will E. Draper, Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA.
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