Feb 11 Prokaryote Gene Regulation PPT-2

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Genes in a chromosome encode proteins or RNA. Examples of RNA include the tRNA and rRNA molecules important for translation, along with many others that we will not explore in this introductory course. The picture on the left reminds you of how where transcription and translation occur in bacteria, while the picture on the right reminds you that the processes of transcription and mRNA processing are carried out in a distinct location (nucleus) from translation (cytoplasm) in eukaryotes.

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In this lecture, we will explore one mechanism used by bacteria to regulate their gene expression. We already looked at one example of how eukaryotes regulate gene expression through the involvement of transcription factors that bind at distal and proximal control elements. We’ll see some similarities in prokaryotes, but also some key differences.

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For bacteria, life is tenuous-their environmental conditions can change at any moment which can result in changes in available nutrients, changes in temperature, or changes in pH. These cells must be able to cope with these changes otherwise they will die. The bacterial chromosome contains almost all of the genes that an organism will need. Since genes largely encode proteins, and the processes of transcription and translation are energy intensive, a bacteria will only express their genes to the extent needed. Therefore, genes have dimmer switches, just like the lights in your dorm room. When the bacteria need more protein, they turn the gene expression up (the dimmer switch slides up to create more light). When they don’t need the protein encoded in a gene, they turn gene expression down to almost immeasurable levels (the dimmer switch slides down to provide almost no light.

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Since transcription and translation are tightly coupled (what is the coupling agent-in other words, what is the intermediate between transcription and translation?) then the cell regulates gene expression at the level of transcription. WHY? In bacteria, genes are arranged on the chromosome in functional units known as operons. We’ll explore these genetic structures in the coming slides, but first you should recall that translation in bacteria starts shortly after translation begins (left hand side of lower figure) and there can be many different ribosomes bound to a mRNA and synthesizing proteins at any given time (top and bottom figures).

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Operons are specific regions in a bacterial chromosome that have multiple genes all regulated together, meaning that they are all either expressed or they are all repressed. There are many operons in bacteria that are related to nutrient utilization. Each operon has several features. There is a promoter region in front (“upstream” or towards the 5’ end) of the first gene, and then a series of genes in series with one another, each one transcribed and translated into separate proteins. The figure shows an operon with three genes, labeled A, B and C. Each one of these genes has its own start and stop codon (green and red segments in mRNA illustrated in figure).

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In an operon, there is an operator DNA sequence (yellow) between the promoter (light green region under the RNA polymerase) and the first gene (gene A in this example). The operator will bind to proteins that block RNA polymerase from moving 5’ to 3’ along the chromosome under specific conditions. We’ll examine a specific operon and its operator in the next section of this lecture.

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Look at the top and bottom scenario and think about the question on the left. We’ll examine this in more detail in class.

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When bacteria like E. coli, a common species in the human gut, are looking for chemical energy, their first choice will often be glucose. In many ways they are just like us—always seeking sugar! When glucose is in low abundance, E. coli must turn on the genes that are needed to use alternative sugars as sources of energy. One of the best studied systems is the Lac operon. Lac stands for Lactose-you learned about this disaccharide in Lecture 3. To use lactose as an energy source, this disaccharide must first be brought into the cell through a channel, called Galactoside permease. Then, lactose must be hydrolyzed to create two monosaccharides—glucose and galactose-through the activity of the enzyme β-Galactosidase. These monosaccharides will be used in glycolysis and other metabolic pathways to generate energy.

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Here’s another view of Lactose utilization by E. coli. When the cell is going to use lactose for energy, it expresses the permease protein and lactose is transported across the cell membrane. β-galactosidase then carries out two different reactions. The first is the hydrolysis reaction we saw on the previous slide to create galactose and glucose (bottom right of figure). The second reaction that β-Galactosidase will carry out is to switch the glycosidic bond in lactose from one form to the other to create the related disaccharide Allolactose.

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Under normal conditions when lactose is not around, there is a regulatory gene known as lacI (pronounced lac – i). The protein that is produced from lacI is known as the Lac repressor. It is present in the cell at all times. In the absence of lactose, the Lac repressor will bind to the operator sequence in the lactose operon, and shut down transcription by blocking the progression of RNA polymerase. NOTE: lacI is regulated separately from the lac operon and it is present in the cell at all times.

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When lactose is present, β-Galactosidase will produce allolactose. The allolactose will then bind to the lac repressor protein and change its quaternary structure. This change in quaternary structure results in the Lac repressor losing its ability to bind to the lac operator DNA sequence. We call allolactose an inducer because its presence allows the expression of the genes in the lac operon (it induces their expression).

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The loss of binding by the Lac repressor results in transcription of the lac operon, and the production of the gene products needed for lactose utilization. Note: The lac repressor is involved in negative regulation! It’s presence will decrease gene expression unless there is enough allolactose to bind to the lac repressor and inactivate it.

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Remember that positive regulation will increase gene expression. The positive regulation of the lactose operon occurs through a protein known as CAP, or Catabolite activator protein. CAP binds to cAMP, a derivative of ATP that is formed in the cell. High glucose results in low amounts of cAMP, while low glucose results in high amounts of cAMP. We’ll see the importance of this relationship on the next slide.

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Here’s CAP and cAMP in action. Note that the cell conditions are: 1) low glucose and 2) high lactose. 1.  Glucose levels drop in the cell and cAMP levels increase. 2.  cAMP binds to CAP and activates CAP to bind to the Lac operon promoter 3.  Active CAP turns on Lac operon transcription 4.  Lac operon transcription results in the utilization of Lactose by the cell.

Think about the following alternatives and make sure you understand why they result in their stated outcomes. 1.  If glucose is low and lactose is low, then there is little expression of the Lac

operon (hint: it relates to the activity of Lac repressor) 2.  If glucose is high and lactose is high, then there is little expression of the

Lac operon (hint: it relates to the activity of CAP) 3.  If glucose is high and lactose is low, then there is little expression of the

Lac operon (hint: both CAP and Lac repressor are involved)

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