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oai:www.repository.cam.ac.uk:1810/2977482021-04-21T20:16:24Zcom_1810_263984com_1810_221767com_1810_256067col_1810_263986

Structural studies of trans-translation 10.17863/CAM.44800 Rae, Christopher Ramakrishnan, Venkatraman Ribosomes translate messenger RNA (mRNA) into protein in all living cells. The faultless production of protein is critical for a vast array of catalytic and structural roles and is essential for the survival of the cell. Ribosomes themselves are made up of both RNA and protein, and are composed of two subunits, each with a separate function. The small subunit reads the mRNA message, directing the large subunit to synthesize a sequence of amino acids to form a protein. In many cases, mRNA may be damaged or truncated in such a way that ribosomes reach the end of the message and become trapped. Rescuing stalled ribosomes is essential as an otherwise lethal build-up of unproductive ribosomes diminishes the translation capacity of a cell. This study focuses on an essential pathway called trans-translation, which resolves stalled ribosomes in nearly all bacteria. Two factors, transfer-messenger RNA (tmRNA) and small protein B (SmpB), form a complex that rescues the ribosome by terminating translation and releasing the ribosome from the mRNA message. In vitro biochemistry in conjunction with cryo-electron microscopy (cryo-EM) was used to visualize frozen snapshots of the ribosome undergoing trans-translation. The structures reveal the coordinated movement of tmRNA and SmpB through the ribosome. Binding interactions between tmRNA-SmpB and the ribosome explain why trans-translation only begins on ribosomes that reach the end of an mRNA and not for actively translation ones. SmpB plays an essential role in positioning tmRNA as together they mimic both a tRNA and mRNA. The movement of tmRNA-SmpB results in a stepwise message swapping from the original mRNA to tmRNA, facilitating the rescue of stalled ribosomes. Overall, this structural study advances our atomic level understanding of the mechanism of trans-translation. University of Cambridge 2019-10-26 Thesis Doctoral Doctor of Philosophy (PhD) en This degree was supported by a Gates Cambridge Scholarship. https://www.repository.cam.ac.uk/handle/1810/297748 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a747a584-624c-4bce-acfb-42b7d9942ae3/download b8603e4a4e5121c619508ec82b8faf38 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/64f79520-0465-4493-8deb-399c1766dc3c/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ translation ribosome trans-translation tmRNA SmpB Cryo-EM