Quantitative dissection of RNA structure formation reveals a cooperative and modular folding and assembly landscape
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Structured RNAs are pervasive in biology with ubiquitous roles in gene expression and regulation. RNAs must fold from a linear chain of nucleotide sequence to attain three-dimensional structure. RNA folding can be described as modular and hierarchical with tiers of structure that form independently: secondary structure forms first and defines the helices followed by formation of tertiary structure. The separation between secondary and tertiary structure is not absolute. Many biological RNAs couple secondary structure changes to RNA tertiary structure formation and link these changes to downstream functional consequences. To predict how these biological RNAs fold requires a deep understanding of the structural intermediates, folding pathways, and mechanisms of cooperativity that promote folding. To test the modularity and predictability of secondary and tertiary RNA folding and assembly, we have investigated the folding and assembly of the P5abc subdomain from the Tetrahymena thermophila Group I intron ribozyme. P5abc folds cooperatively in isolation, binding Mg²⁺ ions and adopting tertiary structure. Mg²⁺ binding is linked to a shift in the secondary structure of seventeen nucleotides and prior work concluded that there is a high degree of cooperativity for this seemingly concerted transition. With the already established principles of RNA modularity in mind, we develop a reconstitution hypothesis to test if cooperative secondary and tertiary folding and assembly of P5abc can be understood from the component pieces. By using rational mutagenesis, we find that higher order folding of P5abc is modular, and we elucidate the physical origins of cooperativity (Chapter 2). With our knowledge of isolated P5abc folding, we demonstrate that the local folding transition within P5abc controls the rate and pathway of assembly with the P5abc-deleted ribozyme core (E[superscript ΔP5abc]), further highlighting the modularity of RNA structure (Chapter 3). Lastly, we show that the kinetics of assembly can be attributed to specific tertiary contacts that form in the assembly transition state such that the rate of a particular folding pathway is dictated by the properties of an individual tertiary contact (Chapter 4). The modularity of RNA structure makes it a reasonable molecule for the origins of life and an adaptable tool for bioengineering applications.