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.
Draper
Will E
WE
Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA.
Hayden
Eric J
EJ
Lehman
Niles
N
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