Our work aims to understand how gene expression is regulated in the chloroplast. In order to achieve this, we seek to develop methods allowing us to determine cryo-EM structures of large multi-subunit gene expression complexes purified directly from native source. Chloroplasts originated from free-living organisms similar to cyanobacteria and, owing to their endosymbiotic origin, they possess their own genome (1). Importantly, the chloroplast genome encodes proteins essential for photosynthesis and, thus, the regulation of their gene expression is critical for plant fitness. These genes are transcribed by a plastid-encoded RNA polymerase (PEP), a 1 MDa multi-subunit complex which is the focus of our project.
It is known that PEP contains an enzymatic core similar to bacterial RNA polymerase, which forms a complex with 12 additional subunits encoded in the nuclear genome. These PEP-associated proteins (PAPs) are not homologous to bacterial transcription factors yet are essential for transcription in the chloroplast and are required for chloroplast maturation (1, 2). Mutants in any of the genes encoding PAPs result in albino plants that are unable to complete their life cycle. Despite their importance, we currently lack an understanding of the biochemical role of PAPs in chloroplast transcription.
To address this knowledge gap, we are optimising a method for the purification of PEP from mustard seedlings. This will enable us to rigorously characterise the PEP complex using an array of biochemical and biophysical techniques, including SDS‑PAGE, peptide mass fingerprinting, mass photometry and cryo‑electron microscopy. Ultimately, an understanding of how PAPs regulate transcription by PEP will give us a more complete picture of how gene expression is regulated in the chloroplast. This will further our comprehension of plant biology, particularly photosynthesis, as well as enable potential applications in chloroplast biotechnology.