Structure and function of the chloroplast transcription machinery

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Plant growth is driven by photosynthesis. Yet how plants produce their photosynthetic proteins is not well understood. The chloroplast contains a genome that encodes key photosynthetic proteins and a unique molecular machinery that expresses them. Despite their importance, how the chloroplast gene expression machinery functions has not been characterised in detail. This project focuses on the first stage of gene expression in the chloroplast, in which genes are transcribed to produce messenger RNAs (mRNAs) that encode photosynthetic proteins. Regulation of chloroplast transcription underpins a key stage of plant development: the maturation of chloroplasts in response to light. This process is observable in the plant turning green and allows photosynthetic proteins to be selectively produced when solar energy is available for them to collect. Yet how chloroplast transcription is activated is not well understood.To advance our understanding of chloroplast transcription and the mechanism of its activation, we will characterise the enzyme that performs this process: the plastid-encoded polymerase (PEP). PEP is a large molecular assembly with at least 16 protein subunits. PEP is remarkable amongst transcription enzymes in that it contains subunits of two evolutionary origins. The core resembles bacterial enzymes and was inherited with the chloroplast genome from a cyanobacterial ancestor. By contrast, the twelve or more proteins that stably bind to the core are encoded in the nuclear genome and imported to the chloroplast. We therefore expect that these proteins, known as PAPs (PEP-associated proteins), orchestrate key regulatory processes unique to the chloroplast.In this project we will visualise the molecular architecture of the chloroplast transcription machinery using cryogenic electron microscopy (cryo-EM). Atomic models of PEP will shed light on how each subunit regulates transcription in response to the needs of the chloroplast. The level of detail provided by modern cryo-EM is immensely valuable to developing new hypotheses, as precise modifications can be designed with predictable changes in activity. We will examine the consequences of making specific changes, using transcription reactions reconstituted with purified components and plant genetic complementation experiments. The outcome will be a better understanding of what role each component of PEP has, how it performs it, and why these processes are essential to chloroplast development and photosynthesis.This project is expected to deepen our fundamental understanding of the biochemical basis of transcription. Decades of detailed study have been performed on the proteins that perform transcription in the eukaryotic nucleus and bacteria. This has shown that collating information about diverse proteins is essential to inferring general principles of how gene expression is regulated. Understanding the unique set of proteins that act on chloroplast genes therefore represents an exciting opportunity to advance this. Transcription regulation is a key component to human health and disease, and this research consequently has diverse potential uses. Photosynthesis has a central role in producing the oxygen and energy that sustains much of life on earth. Detailed structural and biochemical studies on the photosynthetic proteins have revealed in detail how they harness solar energy, and this has provided a valuable foundation for crop improvement and development of diverse biotechnologies. By contrast, equivalent mechanistic studies of the gene expression processes that underpin production of the photosynthetic proteins are largely lacking. This project will answer a complementary set of questions: what determines the timing and level of photosynthetic protein production, and how could we modify this to develop more robust crops and new biotechnological applications?