DNA Regulation PDF

Gene Regulation Prokaryotes & Eukaryotes Controlling 01 the Level of Gene Expression Why is it important to control gene expression? Controlling Gene Expression Genes are not always required to be transcribed and translated. This would be inefficient if all genes were continuously transcribe...

Gene Regulation Prokaryotes & Eukaryotes Controlling 01 the Level of Gene Expression Why is it important to control gene expression? Controlling Gene Expression Genes are not always required to be transcribed and translated. This would be inefficient if all genes were continuously transcribed/translated. Instead, prokaryotes environmental and eukaryotes control their gene expression in response to ___________________________ physiological and _______________________________ conditions. For example, human insulin is only required when the glucose level is high in the blood. Otherwise, insulin is not produced and does not need to be produced. The ideal functioning of an organism requires that genes are turned __________________ “on” and “off” as they are needed. Definitions Gene Regulation: the control and change of gene expression in response to different conditions in the cell or environment. This also determines cell fates. Housekeeping Genes (Constitutive): genes that are expressed all the time, only varying in rate of expression. These genes are required for homeostasis Activators: biochemical molecules that ___________________ promote gene expression. Repressors: biochemical molecules that _____________________ reduce gene expression. Operon: a group of genes clustered together that are regulated by one promoter. 02 Prokaryotic Gene Control Mechanisms What are Operons?  Regulatory Regions of an Operon Regulatory Regions Coding Regions Enhancer Promoter Operator Gene 1 Gene 2 Gene 3 Activates transcription Region that to higher levels RNA regulatory factors Polymerase bind to binding site (ex. repressors) 2.1 Lac Operon What is the Lac Operon? The Lac Operon Lactose is a potential source of energy for prokaryotes that must be obtained from the environment. Prokaryotes use the lactose operon to regulate the gene expression of those proteins required for lactose metabolism. They prefer glucose, but will use lactose when glucose levels are low. The lac operon is a cluster of three genes that code for the proteins involved in the metabolism of lactose. The lac operon consists of a promoter (the site where DNA transcription begins), an operator (the sequence of bases that control transcription), and the coding regions for the various enzymes that actually metabolize the lactose. The CAP binding site is a positive regulatory site that is bound by catabolite activator protein (CAP). When bound, it promotes transcription by helping RNA polymerase bind to the promoter. The Lac Operon Upstream from the operon is a gene that codes for a repressor protein (lac repressor). This protein takes cues from the environment (in this case, the concentration of lactose within a cell) and regulates the production of the lactose-metabolizing proteins. For the lac operon, this protein is called the lacI protein or lac repressor. The genes that code for the lac repressor are always transcribed, so the lac repressor is always present within a cell. The CAP gene is constantly expressed. This means CAP protein is always available in the cell to bind cAMP and "report" glucose levels to the lac operon and other target genes and operons.  When to use the Lac Operon? Prokaryotes (i.e. E. coli) would much rather Two regulatory proteins are involved: break down glucose than lactose. ○ The lac repressor, acts as a lactose To use lactose, the lac operon genes must sensor. be expressed, which encode key enzymes ○ The catabolite activator protein for lactose uptake and metabolism. (CAP), acts as a glucose sensor. To be as efficient as possible, the lac operon These proteins bind to the DNA of the lac should only be expressed when two operon and regulate its transcription based conditions are met: on lactose and glucose levels. ○ Lactose is available ○ Glucose is not available  When to use the Lac Operon? The lac operon is considered to be an Allolactose is inducible operon. known as an It can be turned on inducer: a molecule when the inducer is that triggers present (allolactose), but is otherwise expression of a turned off. gene or operon. When lactose is available, some turn into allolactose in the cell  When to use the Lac Operon? CAP isn't always active Even with lactose (able to bind DNA). It is present and no lac regulated by a small repressor, RNA molecule called cyclic polymerase does not AMP (cAMP). cAMP is a "hunger signal" made bind well to the when glucose levels are mRNA on its own. low. cAMP binds to CAP, allowing it to bind DNA and promote transcription. Without cAMP, CAP cannot bind DNA and is inactive.  Mutation Worksheet DNA Mutation Let’s do some practice to understand DNA mutations better! Mutation Worksheet - Answers  Gel Electrophoresi Video: Gel Electrophoresis s Use these resources or research on your own to learn more about Gel Electrophoresis. Purpose Process Article: Gel electrophoresis (article) | Khan Materials Academy Simulation: Gel Electrophoresis 2.2 trp Operon What is the Trp Operon? The trp Operon Bacteria, such as E. coli, need amino acids to survive. One of these amino acids is Tryptophan. In humans, tryptophan is used to make melatonin and serotonin. Melatonin: helps maintain the sleep-wake cycle. Serotonin: helps with regulating appetite, sleep, mood, and pain. If bacteria can obtain tryptophan from the environment, it will do so. However, if there is no tryptophan available, the E. coli can create their own by using enzymes that are encoded by 5 genes. These 5 genes are found next to each other on the trp operon.  The trp repressor only binds to the operator when tryptophan is present. When tryptophan is around, it The trp operon is a attaches to the repressor molecules repressible operon The trp repressor is encoded by (usually on but can be the gene trpR (not a part of the and changes their shape so they turned off). When trp operon). become active. A small molecule like tryptophan levels are high, tryptophan, which switches a tryptophan interacts with a repressor protein so that repressor into its active state, is it can bind to the operator called a corepressor. and prevent transcription.  With no tryptophan available, the trp repressor is not bound to tryptophan leaving it inactive. While inactive, it cannot bind to the operator, allowing RNA polymerase to transcribe the operon as normal. For the trp operon, the trp repressor acts as both the sensor and switch. It senses if tryptophan is available or not and can accordingly turn the operon “on” or “off”. 03 Eukaryotic Gene Control Mechanisms How are genes controlled through transcription and translation? Eukaryotic Gene Control Mechanisms 1 Transcriptional 3 Translational As mRNA is being synthesized. As the protein is being synthesized. Post-Transcription Post-Translation 2 al 4 al As mRNA is being processed. After the protein has been synthesized. How is DNA packaged? Transcriptional Regulation Regulates which genes are transcribed (DNA to mRNA) or controls the rate at which transcription occurs. Transcriptional Regulation General transcription factors, accumulate on the promoter. They bind to a specific region of the promoter and provide a substrate that the RNA polymerase can bind to. Together, the general transcription factors and RNA polymerase form the transcription initiation complex. This establishes a base rate of gene transcription, which can be further altered by activators and repressors.  Transcriptional Regulation Adding acetyl Adding methyl groups to histones groups to the loosens their cytosine bases in association with the promoter DNA. region inhibits transcription. Acetyl group: (CH3COO–) Methyl group: (-CH3) Transcriptional Regulation An example of the power of gene methylation is seen in agouti mice. Mice whose agouti genes have been turned on can look entirely different in both colour and size, even though they are genetically identical. One mouse may be small and brown, while its twin may be obese and yellow. In normal, healthy mice, the agouti genes are kept in the “off ” position (silenced). Methyl groups are attached to the corresponding regions of DNA, and transcription is prevented. In yellow and/or obese mice, however, the same genes are not methylated. Thus, these genes are expressed or “turned on.” Mice whose agouti gene is “on” have a higher risk of cancer and diabetes. Post-Transcriptional Regulation Post-transcriptional regulation influences gene expression by several mechanisms, including changes in pre-mRNA processing and the rate at which mRNAs are degraded. Examples of of post-transcriptional regulation includes: Alternative Splicing Binding masking proteins to mRNA ○ When bound, mRNA does not undergo protein translation. Changing the rate of mRNA degradation ○ A regulatory molecule, such as a hormone, will directly or indirectly affect the rate of mRNA breakdown. Translational Regulation One important mechanism changes the length of the poly(A) tail of the mRNA molecules. Specific enzymes can add or delete repeating sequences of adenine at the ends of the mRNA molecules. This change in the length of the poly(A) tail may increase or decrease the time that is required to translate the mRNA into a protein. Post-Translational Regulation After mRNA is translated and the protein is produced, it can still be regulated by the cell. Three methods are used: Processing, Chemical Modification, and Degradation. When the protein is produced, it is in an inactive state. Through processing, certain regions of the protein are removed, making it active. During the chemical modification of a protein, certain chemical groups that are attached to the protein are added or deleted, affecting its function. Proteins are subject to degradation as well. Some proteins are used for only a few minutes before they are broken down, while others can last the entire lifetime of an organism. This rate of degradation is under regulatory control, modifying the rate at which the products of gene expression are available. Cancer Can occur when normal regulatory mechanisms are not working/lacking. Mutations within the genome can ultimately lead to cancer. Every exposure to a mutagen has a cumulative effect on the number of mutations. That’s why cancer tends to be more common during old age. A mutation in one cell only passes that mutation to daughter cells, not nearby cells. If a cell does not follow normal cell division, it can produce a mass of undifferentiated cells called a tumour. If this mass of cells grows slowly, remains in place, and does not return once it has been Changes in gene regulation can arise from removed, it is called a benign tumour. If the cells mutations in the promoters, mutations in the grow uncontrollably, invade surrounding tissue, coding regions that affect the functions of the and begin to affect the functions of the organism, protein, or the introduction of foreign DNA from they are called malignant tumours or cancers. viruses. Gel Electrophoresis