טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentInbal Vaknin
SubjectNovel Frontier in Gene Regulation via Expression of
T7 Bacteriophage RNA Polymerase in Yeast
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Assistant Professor Amit Roee


Abstract

Gene expression systems in eukaryotes encompass gene transcription, mRNA editing, translation and post-translational modifications as crucial key steps for protein synthesis. These systems are regulated in nature, yet are inherently noisy. In other words, gene transcription occurs in "pulses", regardless the intensive regulation that may stand behind a given expression system. In particular, synthetic expression systems are no different from the natural systems, but nonetheless can act as simplified models in order to elucidate unknown areas in gene regulation. Therefore, developing such a synthetic expression system embedded in yeast cells, and based on T7 RNA polymerase (T7 RNAP), can potentially carve new paths to study gene regulation. Successful attempts to express the T7 RNAP have been done also in yeast and other higher eukaryotic cells. In addition, it was shown that T7-generated transcripts were accumulating in the cells. However, no protein product was detected, due to the lack of a critical editing step in the target mRNA: G-capping on the 5' end of the mRNA. Hence, a capping enzyme, coupled to T7 RNAP activity, is needed for the translation of the T7-generated transcripts in yeast.

I decided to combine the T7 RNAP and a viral capping complex called D1-D12 capping complex, from the Vaccinia virus, in order to translate the green fluorescent protein (GFP) target gene. D1-D12 complex is a heterodimer enzyme: D1 is the large subunit and is responsible for the mRNA capping, while the small subunit (D12) plays a role as a stabilizer for D1 activity. It was also found out that this complex is a transcription factor in the Vaccinia lifespan.

Two systems (plasmid and integration based systems) were designed in order to investigate the effect on the GFP fluorescence. Also, two 3' untranslated regions (UTRs) for the GFP transcription (i.e. a yeast terminator and native T7 terminator) were tested and compared in order to evaluate the terminators effect on the GFP fluorescence.  

            The current results show that in a synthetic expression system lacking the capping complex, T7 RNAP is expressed and active in the yeast nucleus. Accordingly, high levels of GFP mRNA are detected in the cells. GFP fluorescence levels somewhat elevate, compared to the wild type (WT) strain. However, I hypothesize that the fluorescence will distinctly increase when incorporating the D1-D12 capping complex into the system. Indeed, the GFP fluorescence levels were increased dramatically, especially in cells where the GFP's termination signal is derived from a yeast source. An astonishing finding in this synthetic expression system is the high GFP fluorescence levels when T7 RNAP is absent, while D1 and D12 are expressed in the yeast. This implies that perhaps GFP transcription initiates from a weak yeast cryptic promoter, located somewhere upstream the GFP gene. I suggest that in the presence of D1-D12 complex, the yeast RNAP is stabilized at the cryptic promoter site and GFP transcription may be possible.