Replication of RNA by Transcription Polymerases

• Main Author: Jain, Nimit
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2021
• Subjects: Biochemistry; Chemistry; College professors; Enzymes; Evolution; Evolution & development; Evolution and Development; Medical research; Protocol; RNA polymerase
• ISBN: 9798544205272

While both DNA and RNA can serve as hereditary material in viruses, DNA is considered the carrier of genetic information in cellular life. For RNA to serve as a genetic material in cells, enzymatic machinery to replicate RNA (i.e., synthesize full-length copies of RNA) would be required. However, RNA replicase enzymes that are well-known to replicate RNA have so far only been found in RNA viruses such as influenza and dengue but not in cellular organisms. Interestingly, numerous lines of evidence suggest that two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) replicate their RNA genetic material by redirecting cellular DNA-dependent RNA polymerases (also called transcription polymerases) to serve as RNA-dependent RNA polymerases. This noncanonical RNA replication activity of transcription polymerases could provide a general means for propagating RNA as hereditary material in cellular contexts because transcription polymerases are found across the tree of cellular life. But a molecular-level understanding of the diversity, mechanisms, and origins of RNAs that can be replicated by transcription polymerases was lacking. By integrating next-generation sequencing, microfluidics, bioinformatics, and biochemistry approaches, I have investigated in vitro RNA replication by a model transcription polymerase enzyme from T7 bacteriophage called T7 RNAP. There were three previous papers on RNA replication by T7 RNAP, all published more than two decades ago, which together described five replicating RNA sequences. The origins of the RNAs replicated by T7 RNAP and requirements for RNA replication by T7 RNAP were unclear. I identified hundreds of new replicating RNAs, defined three mechanisms involved in replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rollingcircle synthesis), and described emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the replication of naturally occurring RNA genetic elements and demonstrate how replicating RNAs can originate from non-replicating materials