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New electron microscope images DNA making protein
The mechanism for reading DNA and decoding it to build proteins is common to all animals and plants, but is often hijacked in cancers.
Scientists at The Institute of Cancer Research, London have now captured in exquisite detail, images of molecular machinery in the RNA Polymerase III complex in the act of transcribing/reading a gene. The unprecedented work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), Cancer Research United Kingdom (UK) and the Wellcome Trust, UK.
The discovery of exactly how the molecular mechanism works - published in the journal Nature - could open up new approaches to cancer treatment. RNA Polymerase III is crucial to life in all eukaryotic cells (cells with a cell wall) which includes all animals and plants. In cancer, RNA Polymerase III becomes more active, causing cells to produce larger numbers of the building blocks needed to grow and multiply.
Capturing molecular machinery in the act of binding to DNA, then separating the two DNA strands in preparation to transcribe DNA code can now be observed physically.
Using an advanced form of electron microscopy called Cryo-EM, which was awarded the Nobel Prize in Chemistry in 2017, scientists are able to zoom in and capture images of the transcription reading mechanism with unprecedented detail. Cryo-EM is so powerful it takes pictures of molecules - approximately 5 nanometers or 20,000th of the width of a human hair - at an almost atomic level.
Researchers can now see how components of RNA Polymerase III complex, along with accessory molecules, interact and communicate with each other. The complex reads the DNA code to produce material known as transfer RNA - an essential part of producing protein building blocks needed to allow cells to grow or make new cells. The new knowledge gained from the precise timing and amount of activity transpiring during transcription may lead to new drugs that can split the RNA Polymerase III complex apart by its components.
Cryo-EM involves freezing and imaging samples at -180°C to preserve minute details of protein shapes. This type of microscopy is also an emerging and exciting approach in cancer drug design. Scientists at The Institute of Cancer Research (ICR) have used the technique to reveal five key stages in which the RNA Polymerase III complex reshapes itself to successfully transcribe the DNA code. Now, each of these stages can potentially become the target of new cancer drugs.
Although this molecular mechanism was observed in yeast cells, the same machinery is used in humans.
Cancer cells need large numbers of RNA Polymerase III protein building blocks to grow and divide rapidly, so they become particularly reliant on components of the RNA Polymerase III complex.
"We used a really exciting new type of microscopy called Cryo-EM to do something no scientists have been able to do before. We were able to freeze and catch the RNA Polymerase III complex in the act of attaching to, separating and reading the DNA code. We obtained almost a million independent snapshots using powerful computers to group similar snapshots together, enhancing their detail and obtained a vivid reconstruction of this machinery at work."
RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4–4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation.
Authors: Guillermo Abascal-Palacios, Ewan Phillip Ramsay, Fabienne Beuron, Edward Morris, Alessandro Vannini.
The Institute of Cancer Research, London, is one of the world's most influential cancer research organisations.
Scientists and clinicians at The Institute of Cancer Research (ICR) are working every day to make a real impact on cancer patients' lives. Through its unique partnership with The Royal Marsden NHS Foundation Trust and 'bench-to-bedside' approach, the ICR is able to create and deliver results in a way that other institutions cannot. Together the two organisations are rated in the top four centres for cancer research and treatment globally.
The ICR has an outstanding record of achievement dating back more than 100 years. It provided the first convincing evidence that DNA damage is the basic cause of cancer, laying the foundation for the now universally accepted idea that cancer is a genetic disease. Today it is a world leader at identifying cancer-related genes and discovering new targeted drugs for personalised cancer treatment.
A college of the University of London, the ICR is the UK's top-ranked academic institution for research quality, and provides postgraduate higher education of international distinction. It has charitable status and relies on support from partner organisations, charities and the general public.
The ICR's mission is to make the discoveries that defeat cancer. For more information visit http://www.icr.ac.uk
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Cryo-EM imaging of DNA code being read. Image: The Institute of Cancer Research (ICR), London