In vitro evolution of a processive clamping RNA polymerase ribozyme with promoter recognition

Date created: 
RNA world
In vitro evolution
RNA polymerase
Promoter recognition

The RNA World hypothesis proposes that the early evolution of life began with RNAs that can serve both as carriers of genetic information and as catalysts. Later in evolution, these functions were gradually replaced by DNA and enzymatic proteins in cellular biology. I start by reviewing the naturally occurring catalytic RNAs, ribozymes, as they play many important roles in biology today. These ribozymes are central to protein synthesis and the regulation of gene expression, creating a landscape that strongly supports an early RNA World. Ribozymes have also been produced in the laboratory using artificial rather than natural selection. As phosphoryl transfer reactions are central to the energy balance of all organisms in modern biology, I explore artificially selected kinase, glycosidic bond forming, capping, ligase, and polymerase ribozymes, highlighting the importance of phosphoryl transfer reactions from nucleotide and nucleoside metabolism to the assembly and replication of RNA molecules in an RNA World setting. RNA replicases capable of general and self-replication are thought to have been essential early in evolution. However, how such sophisticated polymerases evolved to enable processive gene expression remains largely unexplored. I performed a complex selective strategy that screened ~10^13 pool variants to isolate a three domain holopolymerase ribozyme, containing a class I ligase catalytic core, an NTP positioning accessory domain, and a processivity clamping domain. This ribozyme uses a sigma factor–like specificity primer to first recognize an RNA promoter sequence, and then, in a second step, rearrange to a processive clamped elongation form. When correctly assembled, the clamped complex results in more than one order of magnitude increase in extension, synthesizing duplexes of 50-107 base pairs in size. The polymerase can also synthesize part of its own specificity primer, programming itself to polymerize from certain RNA promoters and not others, demonstrating how RNA polymerase ribozymes could have preferentially replicated their own genomes and associated genes, while avoiding replicative parasites in a primordial RNA World. The clamp-like mechanism of my selected polymerase could eventually enable strand displacement and improve fidelity, both being critical requirements for replication in the early evolution of life.

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This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
Peter Unrau
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.