Resource type
Thesis type
(Thesis) Ph.D.
Date created
2009
Authors/Contributors
Author: Wang, Qing
Abstract
Ribozyme (catalytic RNA) evolution by in vitro selection is one of the powerful evolutionary systems used to study fundamental mechanisms of molecular evolution. In Chapter 1, I discussed the existence of ribozymes in random pools and showed that step-wise pathways, in which mutation and recombination were fundamental evolutionary mechanisms, were more likely applied to evolve complex ribozymes. In chapter 2, I reviewed some types of mappings between RNA sequence space, shape space and function space, together with some common methods used to discover them. RNA modularity, which was discovered in many natural and artificial ribozymes, was the intrinsic architecture for the evolutions of complex ribozymes by recombination. In Chapter 3, cooperating with Leslie Cheng, I revealed the modularity of B6.61, one of the RNA polymerase ribozymes that might have played critical roles in a putative “RNA World”. We found that the two modular domains of B6.61, the ligase core and the accessory domain, could cooperate in cis, in trans, and when they were hybridized with distinct orientations. We also determined the core motif sequences, the important structure-elements of the accessory domain, and its tertiary interactions with the ligase core by chemical probing, mutagenesis and crosslinking studies. Some three-dimensional models of the accessory domain were then built up for the initial attempt to map the structure-function relationship of B6.61. In Chapters 4 and 5, pre-existing RNA motif sequence(s) were randomly reassembled into nonhomologous random recombination (NRR) pools. In Chapter 4 a NRR pool with a diversity of 108 was generated from a nucleotide synthase ribozyme. The pool contained molecules with insertions, deletions, translocations, and inversions. A following in vitro selection quickly isolated the same functional ribozymes with a broad range of length and led to the rapid unbiased discovery of their core motif by phylogenetic analysis. In Chapter 5, a similar NRR pool derived from eight nucleotide synthase ribozymes was generated and subjected to in vitro selection for RNA ligase ribozymes, resulting in a new but uncharacterized functional RNA. Both studies in Chapters 4 and 5 experimentally demonstrated the power of NRR in shaping the evolution of functional RNAs.
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Language
English
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