Defining the molecular basis of chloroplast transcription of photosynthetic genes
Lead Research Organisation:
University of Oxford
Department Name: Biology
Abstract
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Technical Summary
The first stage in the expression of chloroplast genes that encode photosynthetic proteins is transcription by the plastid-encoded polymerase (PEP) complex. PEP is a regulatory nexus between development and adaptation processes and its subunits are essential to chloroplast biogenesis. Despite its importance, almost no structural information was available for PEP. In recent unpublished work we determined the structure of the intact 21-subunit PEP complex by cryo-EM at a resolution of 2.5 Angstroms. This is the first structural model of the complex, and has provided a wealth of new hypotheses for the role of the PEP-associated protein (PAP) subunits. In addition, this work has provided us a methodological workflow to address key unanswered questions.
In this project, we seek to extend our mechanistic understanding of chloroplast transcription. We propose a multi-disciplinary approach that combines structural information, from cryo-EM and native mass spectrometry, with biochemical analysis of transcription and in plant analysis of protein function.
Our current structural model does not show interactions between PEP and DNA or mRNA mediate regulatory events. We will reconstitute purified PEP on DNA and mRNA in states of transcription initiation and elongation and determine structures using cryo-EM. The regulation of PEP transcription activity will be analysed in vitro, guided by mechanisms known to control bacterial transcription elongation and pausing. We will then test hypothesised mechanisms of regulation and roles of individual PEP subunits that are generated from this structural and biochemical data. This will be achieved by combining in plant and in vitro analyses: a structure-guided genetic silencing-complementation study in Arabidopsis and Nicotiana plants, and recombinant production of PEP mutants for biochemical analysis. Overall, this project will deliver a mechanistic understanding of core chloroplast transcription regulation principles.
In this project, we seek to extend our mechanistic understanding of chloroplast transcription. We propose a multi-disciplinary approach that combines structural information, from cryo-EM and native mass spectrometry, with biochemical analysis of transcription and in plant analysis of protein function.
Our current structural model does not show interactions between PEP and DNA or mRNA mediate regulatory events. We will reconstitute purified PEP on DNA and mRNA in states of transcription initiation and elongation and determine structures using cryo-EM. The regulation of PEP transcription activity will be analysed in vitro, guided by mechanisms known to control bacterial transcription elongation and pausing. We will then test hypothesised mechanisms of regulation and roles of individual PEP subunits that are generated from this structural and biochemical data. This will be achieved by combining in plant and in vitro analyses: a structure-guided genetic silencing-complementation study in Arabidopsis and Nicotiana plants, and recombinant production of PEP mutants for biochemical analysis. Overall, this project will deliver a mechanistic understanding of core chloroplast transcription regulation principles.
