Project Summary
Transcription is a fundamental process that mediates the interplay between genetic information and
phenotypes, thus vital for development, responses to environmental cues, and diseases. Transcription is
primarily catalyzed by DNA-directed RNA polymerases (DdRPs), which are highly conserved among
eukaryotic organisms. It has been well established that regulation from multiple layers controls DdRP-
catalyzed transcription on DNA templates. Interestingly, some DdRPs recognize both DNA and RNA
templates for transcription. This RNA-templated activity regulates gene expression in bacteria and
mammalian cells and is employed by pathogens (i.e., viroids and human hepatitis delta virus) for propagation.
However, the machinery and mechanism for RNA-templated transcription by DdRPs are poorly understood.
Using the replication of potato spindle tuber viroid (PSTVd) as a model, preliminary data showed that a highly
conserved glycine in the second largest subunit of Pol II is critical for DNA-directed but not RNA-templated
transcription, indicating the different requirements of this residue for DNA and RNA templates. In addition,
data showed that TFIIS, a general transcription factor essential for DNA-directed transcription, is dispensable
for PSTVd RNA-templated transcription, suggesting that a specialized group of factors are required for
transcription of DNA versus RNA templates. Furthermore, the eukaryotic transcription factor TFIIIA-7ZF,
which binds RNA but not DNA, has been shown as a critical factor in facilitating Pol II-catalyzed transcription
on PSTVd RNA templates. The TFIIIA-7ZF binding site maps to a region next to the transcription initiation
site and is critical for Pol II binding and PSTVd replication, suggesting that this binding site acts as an RNA
promoter. Together, these findings inspire the central hypothesis that Pol II recruits a specialized group
of factors to selectively recognize RNA as templates and catalyze transcription. Using a novel in vitro
transcription (IVT) assay that is based on Pol II-catalyzed transcription of PSTVd, the goal of this project is to
further dissect the regulatory mechanism for specialized Pol II machinery to transcribe RNA templates. To
achieve this goal, three aims are proposed to further dissect the role of TFIIIA-7ZF in RNA-templated
transcription, map the TFIIIA-7ZF/Pol II binding domains, and characterize the molecular basis of an RNA
promoter. The expected outcomes will provide novel insights into the regulatory mechanism underlying RNA-
templated transcription catalyzed by Pol II and provide valuable knowledge to better delineate RNA promoters.
As this project aims to uncover the basic principles governing the modus operandi of RNA polymerases that
are highly conserved across eukaryotic organisms, the results gained here will provide an in-depth knowledge
of RNA-templated transcription. Such in-depth knowledge will lead to the full understanding of factors and the
regulatory mechanism underlying RNA-templated transcription and facilitate the development of effective
treatments for diseases caused by infectious RNAs.
