114 Understanding Chloroplast Biogenesis: The Role of NCP as an Assembly Factor of the Plastid- Encoded RNA Polymerase Complex in Plants

Faculty Mentor: Chan Yul Yoo (School of Biological Sciences, University of Utah)

Chloroplasts are essential for the survival of all life on earth, as these photosynthetic plastids provide food and oxygen that virtually all living systems rely on. Understanding the molecular mechanisms by which plants develop their chloroplasts is essential for developing solutions to the problems plants experience in the face of climate change. Like mitochondrion, chloroplasts have their own genomes that contain essential genes for chloroplast biogenesis and photosynthesis. In all angiosperms (flowering plants), light initiates chloroplast biogenesis through activation of chloroplast genes by the plastid-encoded RNA polymerase (PEP) holoenzyme complex. Previous genetic screening has identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a key regulator for this light-initiated assembly of the PEP complex. However, the molecular mechanism by which NCP functions for PEP assembly is unknown. I hypothesized that NCP directly interacts with one or multiple of the PEP complex’s subunit proteins to promote the proper folding necessary for its assembly. I used yeast-two-hybrid assays to detect interactions of NCP with each of 16 PEP protein subunits and found that NCP interacts with two of PEP’s core proteins called rpoA and rpoB. These results are consistent with in vivo cross-linked immunoprecipitation coupled with mass spectrometry and in vitro GST pull-down assays. Our results suggest that NCP may function as an assembly factor required for the proper folding as a prerequisite step for the full assembly of the PEP holoenzyme complex. Further investigation is needed to better our understanding of how exactly NCP interacts with these proteins and its role in PEP assembly.