How Does Tobacco Etch Viral mRNA Get Translated? A Fluorescence Study of Competition, Stability and Kinetics
Fluorescence techniques have been used to describe protein-protein and protein-nucleic acid interactions that lead to a competitive advantage for translation of tobacco etch viral mRNA. Using both quenching of intrinsic protein fluorescence and labeling of RNA, equilibrium and thermodynamic parameters were determined to gain insight into preferential binding of protein synthesis initiation factors (eIFs) to tobacco etch virus (TEV) mRNA and the mechanism of binding. Equilibrium data showed that the eIF4F complex binding to TEV mRNA was enthalpically favored and that the complex binds to TEV with greater stability than the cap complex. Kinetic studies using changes in fluorescence anisotropy further characterized the eIF4F-RNA interaction as a bi-molecular, single-step reaction. However, ionic strength dependence of the reaction revealed a possible conformational change after initial binding. These studies provide insight into how viral RNA can successfully compete with host cell mRNA through increasing stability of complexes and kinetic competition.
KeywordsInternal Ribosome Entry Site Association Rate Tobacco Etch Virus Ionic Strength Dependence eIF4F Complex
This work was supported by NSF grant MCB 0814051. The author wishes to thank the many members of her research group who contributed to these studies, in particular Dr. Mateen Khan, Hasan Yumak, Shemaila Sultana, Sibnath Ray, Artem Domashevskiy, Sumeyra Yumak and Dr. John Trujillo.
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