טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLevy Lior
SubjectReconstructing and Discovering New O54 Regulation
Mechanisms in a High Throughput Manner
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Roee Amit


Abstract

Bacterial enhancers are non-translated DNA sequences, which play a fundamental roll in gene regulation and functions as a type of molecular integrator that determines when, where, and how much of a particular gene is expressed. A bacterial enhancer is typically comprised of an upstream activating sequence (UAS) which binds oligomeric activators (also known as enhancer binding proteins- EBPs) that provide the necessary energy for the formation of an open complex on σ54 dependent promoters. Characterization of the relationship between the UAS and its cognate σ54-promoter have been performed previously in low scale and for very specific promoters.

Bacteria can use a variety of mechanisms to regulate the expression of specific genes, a few examples are: (i) competition on the promoter site: in which the concentration change of an inducer or repressor can result in the activation or silencing of specific genes. (ii) Looping based regulation in σ54 dependent promoters: in which DNA binding proteins bound to the looping region can increase or lower the probability of the loop formation and by that control the activation of these kind of promoters. (iii) RNA level regulation: in which secondary structures in the RNA can cause the pausing of an mRNA translation, and (iv) gene regulation by roadblocks and RNA polymerase (RNAP) pausing: in which various DNA binding proteins (e.g. transcription factors or repressors) or even other RNAPs can block the transcription of a trailing RNAP.

I decided to perform a comprehensive characterization of the UAS affinity for EBP’s effect on σ54-promoter activation in enhancer systems, covering all the Ntr regulated promoters with native and synthetic UASs in a combinatorial manner. In addition, I also performed a high throughput experiment using an oligo-library of 12,000 different sequences in order to understand the mechanism behind a silencing phenomenon observed in my experiments and its prevalence in the genomes of E.coli and V.cholera.

My results showed that the σ54-promoter’s activation efficiency is dependent more on promoter’s strength than on the UASs affinity for the EBP. Moreover, I was able to show that it is not possible to predict the UASs affinity for EBP only by the cumulative affinity of its comprising binding sites. In an interesting turn of events my work was also able to 2


show a surprising silencing phenomenon observed using inactivated glnKp promoter and two different upstream promoters (glnAp1 and pLac/Ara). A σ54:RNAP holoenzyme roadblock regulation mechanism was ruled out using site-directed mutagenesis of the glnKp’s sequence and by the comparison of expression using a ΔRpoN (Δσ54) strain, showing that the silencing effect is related to the flanking sequences of the promoter and not to the core consensus sequence. Finally, I was able to show that this silencing phenomenon is widespread in the genomes of E.coli and V.cholera, showing prevalence of 25% in our tested sequences. Future work should be carried out in order to reveal the mechanism/s behind the silencing phenomenon, first focusing on a Shine Dalgarno sequestering mechanism.