Cryo-EM analysis of ribosomal frameshifting

Cell proteins are encoded in DNA, the cell's genetic material, and expressed from so-called messenger RNA copied from DNA. This process of expression involves reading a triplet nucleotide code and translating it into an amino acid polymer, the protein. The process of translation involves structures called ribosomes. These move along the messenger RNA decoding the triplets and adding one amino acid for each triplet to the growing amino acid chain. Viruses that infect cells carry their own genetic material and occasionally, they make messenger RNAs that contain a specific signal (called a frameshift signal) that tells the ribosome to stop making one type of triplet and to start making another. The result is that one messenger RNA can make two proteins. This is typical of viruses; as their own genetic material is usually relatively small, they use all sorts of tricks to maximise the number of proteins they can make. In this project, we wish to examine what happens to the ribosome when it encounters a frameshift signal. The question is 'what happens to the ribosome that makes it misbehave?' To answer this, we will purify ribosomes caught in the act of translating through the frameshift signal and study them by microscopy. The microscopy technique we will use is based on electrons rather than light, so is extremely powerful, and is called cryo-electron microscopy. It is 'cryo' because the samples are frozen in aqueous solution before examination to keep them in a natural and stable state. The images we obtain will hopefully tell us something about how the frameshift signal interferes with the ribosome, and should also be informative about how ribosomes work during normal protein synthesis.

Many viral and some cellular mRNAs contain programmed /1 ribosomal frameshifting signals that instruct the ribosome to change reading frame at a defined point and to continue translation. Frameshift signals are often associated with the expression of virus replicases, for example, retroviral reverse transcriptases. The mRNA signal that specifies frameshifting has two components, a slippery sequence of nucleotides, where the frameshift takes place and an essential stimulatory RNA structure (sometimes a stem-loop but more often an RNA pseudoknot). The mechanism of frameshifting is not fully understood, but likely involves a direct interaction between the stimulatory RNA and the ribosome that perturbs the elongation cycle at the time that the slippery sequence is being decoded. Recently, we succeeded in isolating highly-purified rabbit reticulocyte ribosomes stalled in the act of translating the stimulatory RNA pseudoknot of a coronavirus frameshift signal. In this project, we will use cryo-EM techniques to determine the structure of the stalled 80S-pseudoknot complexes. We will also investigate the structure of ribosomes paused at other stimulatory RNAs, both functional and non-functional in frameshifting. Key macromolecular interactions identified from the cryo-EM reconstructions will be verified by biochemical analysis. The project is a joint venture between a group at Cambridge with experience in the biochemical analysis of ribosomal frameshifting and one at Oxford, with expertise in cryo-EM.