Mechanisms of covalent self-assembly of the Azoarcus ribozyme from four fragment oligonucleotides.
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 MgCl(2). 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.
Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA.
Will E
WE
Draper
Eric J
EJ
Hayden
Niles
N
Lehman
D020627:Azoarcus / Q000201:enzymology*; D007438:Introns; D008957:Models, Genetic; D009154:Mutation; D009843:Oligoribonucleotides / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D017423:Sequence Analysis, RNA
D009843:Oligoribonucleotides; D016337:RNA, Catalytic