Chemistry and Chemical Biology ETDs

Publication Date

Fall 11-14-2016

Abstract

We wanted to develop a system that combines the spatial control of photoactivation and control of translation to build a tool to spatially control translation in neurons. This kind of tool could be used to investigate the role of spatially controlled translation of any protein in neural behavior. In this way the development and growth of neural processes could be studied to elucidate the mechanisms for spatially sensitive events such as pathfinding, repair, or long-term potentiation.

Chemically induced dimerization was used to install a switch into the activation of translation for specific genes. An abscisic acid (ABA) dependent dimerization of the proteins PYL and ABI was engineered to control the proximity of the translation machinery to an RNA sequence by fusing the C-terminus of eIF4G to PYL and fusing the MS2 coat protein to ABI. This produced ABA dependent translation of a reporter gene downstream of the MS2 stem loop. The expression of the reporter protein was low without ABA and increased within hours of addition of ABA.

ABA dependent dimerization of two proteins, PYL and ABI, was engineered to be photoactivatable. ABA was caged with protecting groups, DMNB and DEACM, that can be cleaved by light. This was used to control the activation of biological events with light. We were able to achieve temporal control over the activation of biological activity, but were unable to spatially control this activity.

The photoactivation of ABA dimerization could possibly be used in combination with the activation of translation. This could provide optical control over translation and possibly provide a tool that is capable of studying translation on a sub-cellular scale.

Language

English

Keywords

translation, dimerization, photocleavage, photodimerization

Document Type

Dissertation

Degree Name

Chemistry and Chemical Biology

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Fu-Sen Liang

Second Committee Member

Charles Melancon

Third Committee Member

Lina Cui

Fourth Committee Member

David Peabody

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