Computational Techniques Applied to RNA Structures and Bacterial Genomes



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Modern molecular biology studies increasingly require customized computation support. This is especially true for studies associated with atomic resolution structures and/or NextGen sequencing. In this dissertation, three separate studies are described in which modern computational methodologies play a key role. The first case is a model system that seeks to understand how complexity might arise in a prebiotic RNA World in which RNA polymerization through an RNA replicase was not possible. The experimental system exposes an initial 20-mer seed RNA to 180 min of continuous cycles of synthesis and degradation. The resulting pools of RNA were characterized by NextGen sequencing. The seed RNA rapidly disappeared and was replaced by an increasing number and variety of both larger and smaller variants. The analysis made it possible to characterize the extent and manner in which sequence space was explored. The RNA products lacked large numbers of point mutations but instead incorporate additions and subtractions of fragments of the original RNAs. The system demonstrates that, if such equilibrium were established in a prebiotic world, it would result in significant exploration of RNA sequence space and likely increased complexity. No matter how much complexity can be generated by an RNA World alone, the ability to synthesize peptides would be a momentous discovery. In modern organisms, the PTC, where peptide bonds are fashioned, also serves as an entrance to an exit tunnel so that a growing peptide can leave. In essence, the PTC exit cavity is an evolved RNA nano-pore that accommodates the termini of the A and P-site tRNAs, which carry the activated amino acid or the nascent peptide, respectively. Is this pore unique? In examining various atomic resolution structures, it became clear that such pores can arise readily from the packing of RNA or RNA-like molecules as they increased in size. This hypothesis is supported by our finding of at least five additional pores in the 23S rRNA that range 1.0-1.5 nm in size. Examination of other RNAs identified 11 other RNA pores. Structurally, the PTC and other 23S rRNA pores are very similar, i.e. both are created from the packing of helices, but RNA bases in the latter do not protrude into the pore. Instead, these pores are lined by negatively charged backbone atoms. While the additional 23S rRNA pores seem to lack catalytic function, they illustrate that random RNAs would not have to be very large to create a variety of pores and voids. Thus, pores of the PTC type may have occurred with some frequency in an RNA World, thereby allowing the rapid emergence of primitive translation machinery. Finally, a detailed examination of structural conservation in a ribosomal RNA was undertaken. 5S ribosomal RNA is thought to assist communication between the PTC region of the ribosome and the decoding center on the small ribosomal subunit. This is facilitated by changes in the orientation of 5S rRNA. The canonical 5S rRNA secondary structure includes 30 non-standard base-base interactions. The initial expectation was that these interactions would change as the ribosome went through its synthesis cycle. Forty atomic resolution structures of 5S rRNAs from both different organisms and different stages in the translation cycle were examined. The set of non-standard base pairs continues to be conserved in essentially all of the structures. This strongly suggests that the 5S rRNA behaves as a rod with structural changes originating from the backbone.



Dynamic Combinatorial, Origin of Life, 5S rRNA, PTC, RNA Pores, RNA Sequence Space Exploration, Non-templated Replication, Ribosomes, Early Ribosome


Portions of this document appear in: Tirumalai, Madhan R., Quyen Tran, Maxim Paci, Dimple Chavan, Anuradha Marathe, and George E. Fox. "Exploration of RNA Sequence Space in the Absence of a Replicase." Journal of molecular evolution 86, no. 5 (2018): 264-276. And in: Fox, George E., Quyen Tran, and Ada Yonath. "An exit cavity was crucial to the polymerase activity of the early ribosome." Astrobiology 12, no. 1 (2012): 57-60. And in: Rivas, Mario, Quyen Tran, and George E. Fox. "Nanometer scale pores similar in size to the entrance of the ribosomal exit cavity are a common feature of large RNAs." RNA 19, no. 10 (2013): 1349-1354.