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Title: The c-myc IRES : structure and mechanism
Author: Le Quesne, John P. C.
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2000
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The proto-oncogene c-myc is central to the process whereby the cell commits itself to quiescence, differentiation, proliferation of apoptosis, and the expression of Myc protein is controlled at several levels, including translation. The 5' UTR of c-myc has been shown to contain an internal ribosome entry segment (IRES), allowing translation to proceed via an internally initiated mechanism. To determine the secondary structure of the IRES, structural data were obtained by chemical probing of 5' UTR RNA in vitro. These data were used as constraints upon the "mFold" RNA secondary structure prediction algorithm, and the model was refined by phylogenetic analysis. The resulting model contains a number of interesting features. There is no detectable structural homology with viral IRESs. Mutations were introduced to determine the importance of various IRES moieties. Surprisingly, the IRES seemed resistant to relatively gross structural changes, and a number of mutations were seen to significantly activate IRES function, suggesting that the IRES is in a state of constitutive repression. The point at which the ribosome enters and begins scanning was investigated, revealing that entry occurs in an unstructured region of the IRES, upstream of an inhibitory pseudoknot element that must be disrupted before ribosome entry can occur. It has previously been noted that the c-myc IRES fails to function in RRL in vitro translation assays. In order to obtain an in vitro assay to aid isolation of specific trans-acting factors, several cellular extracts were tested for their ability to stimulate IRES activity in vitro. Nevertheless, the IRES was not activated in vitro. From these data, a picture of the c-myc IRES that is distinctly different from the viral paradigms has emerged, and a model of the IRES mechanism is presented and discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available