The photosynthesis of plants and their eventual production of oxygen and chemical energy with the help of sunlight is essential for life on Earth. For the first time, researchers from Göttingen and Hanover have now succeeded in visualising the copying machine of chloroplasts, the RNA polymerase PEP, in high-resolution 3D. The detailed structure provides new insights into the function and evolution of this complex cellular machine, which plays a key role in reading the genetic instructions for photosynthesis proteins.
With PEP to energynergie
"A unique molecular copying machine, an RNA polymerase called PEP, reads the genetic instructions from the genetic material of the chloroplasts", Professor Hauke Hillen explains, research group leader at the Max Planck Institute (MPI) for Multidisciplinary Natural Sciences, professor at the University Medical Centre Göttingen and member of the Göttingen Cluster of Excellence ‘Multiscale Bioimaging’ (MBExC). In particular, it is essential for activating the genes required for photosynthesis, emphasises Hillen. Without a functioning PEP, plants cannot photosynthesise and remain white instead of turning green.
Both the copying process and the copying machine itself are complex: It consists of a multi-part basic complex, whose protein subunits are encoded in the chloroplast genome, as well as at least twelve attached proteins, called PAPs. The nuclear genome of the plant cell provides the blueprints for these. 'So far, we succeeded in structurally and biochemically characterising a few individual parts of the chloroplast copying machine, but we lacked a precise insight into its overall structure and the functions of the individual PAPs,' explains Prof. Dr Thomas Pfannschmidt, Professor at the Institute of Botany at Leibniz Universität Hannover.
Detailed 3D snapshot
In close collaboration, researchers led by Hauke Hillen and Thomas Pfannschmidt have now succeeded for the first time in visualising a 19-part PEP complex in 3D with a resolution of 3.5 angstroms - 35 million times smaller than a millimetre.
"We isolated intact PEPs from white mustard, a typical model plant used in plant research", explains Frederik Ahrens, team member in Pfannschmidt's group and one of the first authors of the study now published in the journal Molecular Cell. The scientists then used cryo-electron microscopy to create a detailed 3D model of the 19-part PEP complex. To do this, the samples were shock-frozen ultra-fast. The researchers then photographed the copying machine thousands of times and down to atomic level from a wide variety of angles and combined them into an overall image using complicated computer simulations.
A deeper understanding of the evolution of photosynthesis
The research collaboration also used databases to search for evolutionary clues. Their goal was to find out whether the observed architecture of the copying machine can be transferred to other plants. These new findings on the copying process of chloroplast DNA contribute to a better understanding of the fundamental mechanisms of the biogenesis of the photosynthesis machinery. In future, they could potentially also be utilised in biotechnology.
The study was funded by the German Research Foundation (FOR2848, SFB1565, PF323-7 and SPP 2237 MadLand (PF323-9)) and as part of the Excellence Strategy (EXC 2067/1 - 390729940) via the Cluster of Excellence ‘Multiscale Bioimaging: From Molecular Machines to Networks of Excitable Cells’ (MBExC) as well as by the European Research Council (ERC) as part of the EU Horizon 2020 programme with the ERC Starting Grant MitoRNA (Grant agreement no. 101116869).
