Theses and Dissertations

ORCID

https://orcid.org/0000-0001-9270-8097

Issuing Body

Mississippi State University

Advisor

Wang, Ying

Committee Member

Gordon, Donna M

Committee Member

Thornton, Justin A

Committee Member

Popescu, Sorina C

Date of Degree

12-9-2022

Document Type

Dissertation - Open Access

Major

Biological Sciences

Degree Name

Doctor of Philosophy (Ph.D)

College

College of Arts and Sciences

Department

Department of Biological Sciences

Abstract

Transcription is a fundamental process catalyzed by DNA-dependent RNA polymerases (DdRPs). Interestingly, some DdRPs can use both DNA and RNA as templates for transcription. This RNA-dependent RNA polymerase (RdRP) activity of DdRPs is used by RNA-based pathogens such as viroids and hepatitis delta virus for replication. In addition, RdRP activity of DdRPs widely occurs in various organisms to regulate gene transcription. Despite the importance of this intrinsic RdRP activity of DdRPs, associated factors and mechanisms are in their infancy stage. We employed potato spindle tuber viroid (PSTVd) as a model to study RNA-templated transcription. Here, we present evidence showing that circular PSTVd templates are critical for the synthesis of longer-than-unit-length (-) strand products. Further, we show transcription factor IIS is dispensable for PSTVd replication supporting de novo transcription on PSTVd RNA templates. The absence of canonical general transcription factor, TFIIS from PSTVd-templated transcription complex led to the hypothesis that RNA-templated transcription has a distinct organization on the RNA template. To test this hypothesis, we used our well-established in vitro transcription (IVT) system and demonstrated that RNA polymerase II (Pol II) accepts minus-strand for transcription. In addition, transcription factor TFIIIA-7ZF is needed to aid Pol II transcription activity. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed that the first three zinc finger domains are pivotal for template binding. Notably, we identified a remodeled Pol II complex for viroid transcription that is missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9. General transcription factors for DNA-templated transcription are also absent in the transcription complex on the RNA template. This remodeled Pol II complex still possesses the transcription activity on PSTVd RNA template. Collectively, our data illustrate a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

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