Year: 2019
Type: dissertation
Abstract: The precise spatiotemporal regulation of protein synthesis (translation) is essential for many complex biological processes such as memory formation, embryonic development and tumor formation. Current methods for studying protein synthesis offer a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis non-invasively within minutes and with a spatial scale as small as a single synapse. This thesis presents several novel optogenetic constructs designed to control protein synthesis via well-known binding interactions of the translation initiation machinery. Chapter 2 presents a yeast system capable of screening optogenetic designs for the light-dependent down-regulation of protein synthesis. Using this system, we discovered cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of the LOV2 domain from Avena sativa (AsLOV2) and 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. This construct was improved, producing cLIPS2. We show that these constructs can inhibit translation in yeast harboring human eIF4E and bind human eIF4E in vitro in a light-dependent manner. Chapter 3 presents the characterization of LITRs (LOV2 insertion in Translational Regulators), a group of constructs where AsLOV2 is inserted into flexible loops of eIF4E (eukaryotic initiation factor 4E). With a different yeast-based screen, we discovered LITUP1, a light-dependent up-regulator of protein synthesis. We showed that the linker sequences between AsLOV2 and eIF4E were coupled to the activity of LITUP1. This led to the discovery of LITUP2, an improved version, and LITDOWN1 a light-dependent down-regulator of protein synthesis. Chapter 4 presents two photocaging strategies using Dronpa and RsLOV. These were initial attempts at designing optogenetic 4EBP2 and eIF4E constructs, which encountered difficulties with in vitro characterization. Altogether, the constructs presented in this work expand the current optogenetic toolbox and will hopefully allow researchers to dissect the dynamic molecular events underlying translation.
Source: TSpace (University of Toronto)
Author: Huixin Lu
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3093143619