BioM2 Recombinant DNA-based molecular techniques 2020-2021 part II analysis of transcriptional regulatory sequences and protein-protein interaction.pdf

Recombinant DNA-based molecular techniques (part II) Analysis of DNA regulatory sequences and protein-protein interaction Prof. Mamoun Ahram Analysis of transcriptional regulatory sequences: Role of enzymes What are transcriptional regulatory sequences? Firefly luciferase Luciferase reporter assay P...

Recombinant DNA-based molecular techniques (part II) Analysis of DNA regulatory sequences and protein-protein interaction Prof. Mamoun Ahram Analysis of transcriptional regulatory sequences: Role of enzymes What are transcriptional regulatory sequences? Firefly luciferase Luciferase reporter assay Purpose: study the activity of a gene at certain conditions or elucidate the function of certain regions of the promoter Only the regulatory region (e.g. promoter, PPE, etc.) of the gene is placed upstream of a “reporter gene” such as the luciferase gene in a plasmid. The plasmid is transfected (inserted) into cells, and the expression level of luciferase (instead of the original gene itself) is measured. Example The promoter with deleted regions The complete promoter Any good promoter (positive control) No promoter (negative control) Repressor region Activating region Core Protein-protein interaction Co-immunoprecipitation Yeast two-hybrid system starting from a DNA library Proteins form complexes (Co)-Immunoprecipitation Antibody molecules that target a specific protein are conjugated to special beads. A mixture of cell proteins are added to the beads. Only the protein of interest is precipitated as well as other proteins bound to it (co-precipitated). What is a DNA library? A library can be created for DNA fragments just like book libraries. You can have clones of bacteria each containing a specific piece of DNA. You can save these clones in the freezer and take whichever clone you want to study. http://www.sumanasinc.com/webcontent/animations/content/dnalibrary. html Genomic vs. cDNA libraries How? Genomic vs. cDNA libraries Yeast two-hybrid system Taking advantage of domains In yeast, an upstream activating sequence (UAS) exists. UAS is controlled by a transcription factor that is made of two domains A DNA-binding domain (BD) An activation domain (AD) that is responsible for the activation of transcription. Both must be close to each other in order to transcribe a reporter gene such the LacZ gene. https://www.youtube.com/watch?v=okxle_hTaZ0 https://www.youtube.com/watch?v=NxNfibcNk_Y Production of a recombinant protein Gene A Domain Gene B Domain Protein A Domain Domain Protein B Recombinant gene Recombinant protein Quick illustration Cloning of hybrid proteins In order to discover unknown proteins (Y’s) that interact with a known protein (X), the X gene is cloned so it is produced recombined with the DB domain and the unknown Y gene (or genes) are separately cloned so that they are produced recombined with AD. Both recombinant plasmids are transferred into yeast cells so all of them express the known X gene-BD hybrid, but each one expresses a different unknown Y gene-AD hybrid. Why is the LacZ gene used? What is X-gal? Yeast cells are grown in the presence of a lactose analog called X-gal, which generates a blue product when cleaved. When the LacZ gene is activated, beta-galactosidase is produced, which cleaves X-gal generating blue colonies. X-gal The possibilities and outcomes Blue yeast colonies are picked and plasmids are isolated to identify the unknown genes/proteins that interact with the known gene/protein.