Emerging evidence indicates that mRNA synthesis and breakdown are tightly coupled processes. Although most proteins act at only a single step in the mRNA life cycle, the multi-subunit Ccr4-Not complex controls gene expression at all stages. A recent study also suggested that Ccr4-Not may physically interact with RNA polymerase II (Pol II). To investigate this, scientists of the EU-funded POL2-CCR (Structure-function analysis of mRNA metabolism by Ccr4-Not) project set out to perform structure-function analysis of Pol II-Ccr4-Not, using cryo electron microscopy (cryo EM). Initially, they optimised the cryo EM workflow for Pol II samples to improve resolution and image quality. Specialised software was used to generate well-defined, high-resolution Pol II structures without reconstruction artefacts. After screening various Ccr4-Not complexes for Pol II binding, researchers found that the binding interface was located in the nuclease module of the complex. In addition, they tested different potential substrate mRNAs for their capacity to stabilise the three-dimensional structure of the Ccr4-Not Pol II complex and facilitate high-resolution structure determination by cryo-EM. However, the density of Ccr4-Not on Pol II was insufficient to produce interpretable images. The next step is to generate more biochemical and functional data to allow the design of better suited mRNA Ccr4-Not Pol II complexes. Project partners also investigated the mechanism underlying the coupling of the transcription and translation processes in bacteria. Cryo-EM images unveiled the structure of the transcription-translation complex and identified Pol II position as well as the critical regions for interaction. This allowed delineation of the mechanism for direct gene expression control by coordinated transcription and translation. Collectively, the POL2-CCR study results showed that transcription-translation coupling is a conserved feature, which coordinates gene expression and allows bacteria to respond to environmental clues and cellular signals.
mRNA, Ccr4-Not, RNA polymerase, POL2-CCR, cryo electron microscopy