|M.Sc Student||Cohen Roni|
|Subject||Understanding Regulation of Translation through RNA|
Structure and Investigating Regulatory Synthetic
Long Non-coding RNA(slncRNAs)
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Roee Amit|
|Full Thesis text|
We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein−RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (−13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qβ (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2′-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qβ to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qβ-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional−post-transcriptional circuit can be utilized as a binding assay to quantify RNA−RBP interactions in vivo.