Synthetic Evolutionary Origin of a Proofreading Reverse Transcriptase | AIChE

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Synthetic Evolutionary Origin of a Proofreading Reverse Transcriptase

Authors 

Ellefson, J. - Presenter, University of Texas at Austin
Ellington, A., University of Texas at Austin

While proteins involved in fundamental biological processes are under extraordinary functional constraints, recent work has shown that core components in the central dogma can be engineered to have radically altered properties, such as the creation of expanded genetic alphabets, tethered ribosomes, and rewiring of the genetic code.  One of the last discovered components of the central dogma, reverse transcriptase (RT), has for the most part been the province of a single family of enzymes of ancient evolutionary origin and are inherently error prone due to their lack of a proofreading (3'- 5' exonuclease) domain. To determine if the lack of proofreading is a historical coincidence or a functional limitation of reverse transcription, we attempted to  evolve a high fidelity, thermostable DNA polymerase to efficiently use RNA templates. The Reverse Transcription Xenopolymerase (RTX) is a new, distinct evolutionary lineage that maintains the DNA proofreading activity of its parent with a RNA template, which was shown to greatly improve RT fidelity. Mutations in highly conserved molecular checkpoints render RTX capable of polymerizing either DNA or RNA templates with similar efficiencies, enabling applications such as single enzyme RT-PCR and direct RNA sequencing without cDNA isolation. The creation of RTX confirms proofreading is compatible with reverse transcription and reemphasizes that core molecular machinery that has otherwise been conserved over billions of years can be engineered to attain novel functionality. 

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