Molecular BioChemistry II PDF

2 Asma’a Abu-Qtaish Farah Saleh Mamoun Ahram Analysis of DNA regulatory sequences and protein-protein interaction In this sheet, we will discuss the places where we can use the techniques we learned in our previous lecture to analyze DNA regulatory sequences and investigate protein-protein interacti...

2 Asma’a Abu-Qtaish Farah Saleh Mamoun Ahram Analysis of DNA regulatory sequences and protein-protein interaction In this sheet, we will discuss the places where we can use the techniques we learned in our previous lecture to analyze DNA regulatory sequences and investigate protein-protein interactions. ❖ Analysis of transcriptional regulatory sequences (Role of enzymes): Here we will talk about an enzyme that we can use to investigate transcriptional regulatory sequences, but first, let's take a look at fireflies and the beautiful light they produce. Then, we'll figure out the rest. Scientists were fascinated by the light emitted by fireflies from their tails. Upon studying this phenomenon, they discovered that fireflies contain D-luciferin, which is converted into oxyluciferin (the molecule responsible for producing light) by an enzyme called firefly luciferase, similar to how GFP functions in jellyfish. Scientists are smart and incredibly creative, they took advantage of this enzyme and used recombinant DNA technology to study the activity of a gene at certain conditions or elucidate the function of certain regions of the promoter which is the purpose of “Luciferase reporter assay”. Reporter: is a gene that provides a measurable signal indicating the activity of a specific region inside the cell under certain conditions, or the importance of several regions in regulating gene expression. Before we get into “Luciferase reporter assay” we have to know the transcriptional regulatory sequences. This is the eukaryotic gene, with a transcription start site (+1 site), exons (the protein-coding regions of the gene), and introns (which are spliced out to form mature mRNA). Today, our focus is on the non-coding sequences that regulate gene activity. These include the TATA box (a basal promoter region), the promoter proximal element (sequences like operons), enhancers (sequences that enhance gene expression), and silencers (sequences that inhibit gene expression). → What do we do in “Luciferase reporter assay”? First, we put the promoter that will control the transcription of the Luciferase gene ahead of it. In this setup, the gene of interest is not our focus; instead, we are interested in the regulatory element that controls the expression of that gene (we will discuss the role that the regulatory region plays after this topic). Cells are not selective about the gene they transcribe; they only care about the regulatory element. If the regulatory element binds to the gene, the cell will express it. After placing the promoter ahead of Luciferase, we transfect it into the cell. Here, proteins control the expression of the luciferase gene instead of the gene of interest. If the gene is expressed at a high level, then luciferase will also be expressed at a high level, and vice versa. From the slides Next, the sample is subjected to certain conditions, and the amount of light produced by the cell is measured. This measurement indicates which conditions actively or negatively regulate the expression of the gene of interest, based on the level of luciferase expression. → Why do we care about the regulatory region? Regulatory regions play a crucial role in determining the amount of a gene that will be transcribed. 3 - No promoter (Negative control): Without any promoter, there will be minimal or zero expression of Luciferase. - Any good promoter (Positive control): This is a strong promoter that will give us the maximum expression of Luciferase, this promoter is not specific to the gene of interest.. 2.1. The regulatory sequence of the gene of interest: The transcription starts site - The complete promoter: This is the actual promoter that normally transcribes the gene of interest. - The promoter with deleted regions: Here, we observe three states: 1. Removing parts of the repressor region leads to increased expression of luciferase. This indicates the presence of a repressor region in the promoter. 2. Removing parts of the activator region results in decreased expression (drop) of luciferase. This suggests the presence of an activator region in the promoter. 3. Removing the core itself leads to no expression of luciferase, returning to the negative control state. The Core Promoter: The RNA polymerase binding site that produces the basal (minimal/standard) expression of a certain gene. ❖ Protein-protein interaction - Proteins don’t act by themself, they interact with other proteins in order to produce an effect on cells. - Here is an example of protein interactions: a protein with two domains, one at the N-terminus and the other at the C-terminus. - The N-terminal domain interacts with two other proteins, which can also interact with each other. - The C-terminal domain can interact with four different proteins. However, the proteins located at the side do not interact with each other. - Studying such complex protein-protein interactions requires techniques like Coimmunoprecipitation1 and the yeast two-hybrid system2. 1. 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, in Coimmunoprecipitation, we pull down not just the protein of interest but also all other proteins that interact with it. - After removing the nonbinding proteins, we isolate the antibody-protein complex for further study using techniques such as immunoblotting (western blot), SDS-PAGE, and others. Remember: Southern blot → for DNA Northern blot → for RNA Western blot → for proteins → 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 from catalogs that indicate their location. To know more about DNA library watch these videos: https://www.sumanasinc.com/webcontent/animations/content/dnalibrary Genomic library cDNA library A genomic library contains all the genetic material of an organism, including exons, introns, noncoding regions, and more, without removing any part. - a cDNA library only contains exons, as it is derived from mRNA using reverse transcription. - it's cleaner and simpler If we create genomic libraries from different cells, those libraries would be the same because the genome is identical in skin, nerve cells, etc. if we create cDNA libraries from different cells, those libraries would be different because the genes expressed in skin cells are distinct from those expressed in nerve cells. They represent active genes in a cell. - Both libraries are created by cutting the DNA with restriction endonucleases. - After cutting, the DNA fragments are recombined with a plasmid using ligase. - The recombinant plasmid is then introduced into bacteria, where it replicates and produces a clone. 2. Yeast two-hybrid system (starting from a DNA library) - Taking advantage of domains, in the previous sheet, we discussed the advantages of protein domains and how we can recombine them while still maintaining their function and structure. Recap of domains: - In yeast, an upstream activating sequence (UAS) exists, it’s the binding site of Gal4 - 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. Gal4 is a transcription factor that induces the transcription of a gene when it binds to the UAS. Quick illustration For more understanding watch these videos: https://www.youtube.com/watch?v=okxle_hTaZ0 https://www.youtube.com/watch?v=NxNfibcNk_Y Using recombinant DNA technology and genetic engineering we will have special yeast cells that have a reporter gene under the control of UAS and Gal4 factor. The yeast two-hybrid system is used to investigate protein-protein interactions. In this method, two hybrid proteins are created: One consists of a DNA-binding domain from the Gal4 transcription factor fused to one protein of interest, and the other contains an activation domain fused to a second protein of interest. If these two proteins interact, the Gal4 DNA-binding domain and activation domain will be in close proximity, leading to the transcription of a reporter gene such as LacZ. If the proteins do not interact, the two domains will not be close enough for transcription to occur. Cloning of hybrid proteins Known - 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 BD and the unknown Y gene (or genes) are separately cloned so that they are produced recombined with AD. Unknown - 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 (Y1, Y2,… etc). → 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 (AD and BD get close to each other), beta-galactosidase is produced, which cleaves X-gal generating blue colonies. → The possibilities and outcomes: 1. 1. Yeast cells producing beta-galactosidase with the normal Gal4 having both the DNA-binding 2. domain (BD) and activation domain (AD) together → will produce blue colonies 3. 2. Yeast cells expressing only the DNA-binding domain (BD) → will not result in any transcription. 4. 3. Yeast cells producing only the activation domain (AD) (the Y with the AD) → serve as a negative control and will not produce blue colonies. 4. In the experimental sample containing X (the known protein) with Y (the protein being tested): - if X and Y interact → colonies will appear blue. - If X and Y do not interact → colonies will not be blue. - Blue yeast colonies are picked and plasmids are isolated to identify the unknown genes/proteins (Y) that interact with the known gene/protein (X) using PCR for example. THE END OF SHEET #2