Cryo-electron Microscopic Studies of Ribosomal Complexes with Biomedically Important Viral mRNAs

Abstract

Some viral mRNAs, such as from Hepatitis C virus, hijack cellular translational machinery by binding directly to the ribosome and circumventing the need for cellular initiation factors. They accomplish this through structured elements within the mRNAs called internal ribosome entry sites (IRESs). Participants of this course will visualize ribosomes in complex with viral IRESs at high resolution.

Objective

The goal of the course is to acquire the most important techniques and methods for the purification and structural characterisation of macromolecular complexes by transmission electron microscopy. The emphasis of the course is on the special practical requirements for the application of these techniques on macromolecular structures in the MDa range.

Content

Protein synthesis is a very energy intensive process that can consume over half the total metabolism of a cell. In eukaryotes, translation is therefore tightly regulated at the stage of initiation. Regulatory processes are much more complex at this step than in prokaryotes and a large number of RNA modification processes and translation initiation factors are required to ensure faithful initiation, elongation and termination of translation. Viral messenger RNAs are often produced by their own machinery, however, and need to be incorporated into the host translation machinery without the usual processing and therefore many viruses have developed strategies to circumvent the need for initiation factors. They accomplish this through highly structured elements within their RNA called internal ribosome entry sites (IRESs) that are able to initiate translation without the normal signals. Some viral IRESs, such as the IRESs from polio-virus or HIV, require most of the normal eIFs and even additional proteins. Others, such as the hepatitis C virus IRES, are able to bind directly to the ribosome and need only a few of the normal initiation factors. Within the Ban lab, we have studied, and continue to investigate, medically relevant viral IRESs. The course will involve producing, and attempting to determine the structures of, IRESs that have yet to have had their ribosome-bound structures resolved. A variety of purification techniques, including preparative gel electrophoresis and ultracentrifugation, will be used during the purification of macromolecular complexes. Purified assemblies will be then investigated functionally. Students will then characterise their samples structurally through transmission electron cryo-microscopy (cryo-EM), including sample preparation, microscopy, data evaluation and the calculation of densities. Finally, students will learn how to build and refine molecular models into parts of the calculated cryo-EM density. The participants will be working on a closed project related to current research within the laboratory and throughout the course the practical work will be accompanied by brief theoretical introductions. The principal aim of the course is to strengthen the skills required to independently conduct meaningful biophysical and biochemical experiments and to provide an early introduction into the structural characterisation of cellular macromolecular assemblies.

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