Open Access Dissertation
Date of Award
V. Hari Ph.D.
The CI protein of tobacco etch potyvirus is known to be an NTPase as well as a putative RNA helicase. In a previous study, the CI gene and three mutants were constructed and expressed in the E. coli expression vector pET 11a. In this study the TEV CI gene was cloned into E. coli expression vector pET 15b and five other mutants were constructed. These constructs in pET 15b, along with the pET 11a constructs, were sequenced to verify the mutations made and the reading fram of the protein. The pET 15b constructs were expressed and assayed for ATPase, ATP binding (only those proteins that were ATPase activity deficient) and RNA binding activities. The pET 11a constructs were expressed and assayed for ATP binding (only those proteins that were ATPase activity deficient) and RNA binding activities. Experiments on the bacterially-expressed CI protein and its mutants showed that (1) the non-domain N-terminal regions of the CI protein (sequences up to domain I) are dispensable for both ATPase activity and RNA binding, (2) that domain I is necessary for ATP binding and ATPase activity, but it alone is not sufficient for ATPase activity, (3) that domains lA and VI are both RNA binding domains and that the presence of one or the other is sufficient for RNA binding. In addition this study showed that the CI protein expressed in virus-infected plants is glycosylated and tyrosine-phosphorylated. This observation is important since it leads to speculations that the CI protein may have roles in signal transduction, membrane transport and other biological functions.
Browning-Kelley, Mary Evelyn, "Mutational analysis of the tobacco etch potyvirus cylindrical inclusion protein" (1998). Wayne State University Dissertations. 1240.