Regulation of Gene Expression Chapter 18

Warm Up Explain the difference between a missense and a nonsense mutation. What is a silent mutation?

Regulation of a Metabolic Pathway Feedback Inhibition- inhibits the activity of the first enzyme in the metabolic pathway. Regulation of Gene Expression- enzymes are controlled at the transcription level, by turning genes on/off. Each cell type contains the same genome, but expresses a different subset of genes. (Ex: the genes that code for eyes, don’t express hair). E.Coli must have tryptophan to survive. They try to conserve energy typically by obtaining trp from the host. When levels are absent/low, they begin to synthesize their own. This gene expression is regulated through 2 different mechanisms 2) The cell stops making the enzymes that catalyze the syntheses of tryptophan. In this case, it happens at the transcription level. The synthesis of messenger RNA coding for these enzymes. Switching off many of the genes in this metabolic pathway. Gene expression is controlled by operons- discovered in 1961 by Jacob and Monod. The five genes that code for these enzymes are clustered together on the bacterial chromosome. A single promoter serves all five genes, which together is a transcription unit. Thus transcription of this gene actually produces 5 polypeptides that make up the enzymes in the tryptophan pathway. The cell can translate this into 5 different polypeptides because the transcription unit is punctuated with 5 different start and stop signals. When enzymes that function together are coded for side by side in the genetic sequence, it makes it easier to turn them on or off in the promoter region. When tryptophan is needed, all of the enzymes needed to make it are turned on and synthesized at one time. This is called coordinate control.

Operons Operator- a segment of DNA that controls the access of RNA polymerase to the genes. Found within the promoter sequence. (on/off switch) Operon- the operator, promoter, and genes they control- the entire stretch of DNA required for metabolic pathway. (trp operon) Described by Jacob and Monod The on/off switch for a particular segment of DNA is called the operator. Found in between the promoter and the exons. Naturally this is switch is turned on, and RNA polymerase is allowed to bind to the promoter sequence. It can be switched off by a protein called the trp repressor.

Types of Operons Repressible Operon- usually on but can be inhibited. (Ex: trp operon) Inducible Operon- usually off but can be stimulated. (Ex: lac operon) *Both are examples of negative gene regulation. Both are inhibited or stimulated when a small specific molecule interacts with a regulatory protein. Lac = lactose, this is what Jacob and Monod first did their research on. They inhibit gene function in some ways.

Repressible Operons Repressor- protein that can turn off the operon by binding to the operator and blocking the attachment of RNA polymerase, preventing transcription of the genes. Regulatory Gene- codes for a repressor or similar protein that controls the transcription of another gene or group of genes. Corepressor- a small molecule that cooperates with a repressor protein to switch an operon off. Promoter includes TATA box which RNA polymerase recognizes. This is the mRNA strand. Previously, there were other introns between the observed exons. A repressor protein is specific for the operator of a particular operon. The repressor that turns off the trp operon has no affect on the other operons in the E. coli. The repressor is coded by its own gene, called the regulatory gene (trpR) which is located upstream from the trp operon and has its own promoter. Regulatory genes are expressed continually at a low rate, leaving a few repressor molecules always present in cells. How then, is the operon not switched off permanently? These repressors have two forms: active inactive, without their corepressor, (Which in this case is the tryptohphan molecule) then they are inactive- so having a few in the cell will not stop production. When tryptophan accumulates in the cell, tryptophan binds with the repressor, which activates it- and it binds to the operator, turning the switch off, and inactivating the transcription unit that produces tryptophan. When trp is needed, it is pulled from the repressor, which reactivates the operator for trp production.

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Inducible Operons Inducer- a small specific molecule that binds to the repressor which inactivates it. Lactose is available to E.coli when the host drinks milk. Lactose (a disaccharide) is then broken down into its 2 sugars, galactose and glucose- with the help of an enzyme Beta-galactosidase. Typically only a few of these enzymes are available in the cell in the absence of lactose. But as lactose is added to the environment the enzyme increases a thousand-fold in about 15 minutes. The gene for B-galactosidase is part of the lac operon, which includes two other genes for enzymes that function in lactose utilization. All coded in one transcription unit with the same operator and promoter. The regulatory gene, lac I is located outside of the operon and codes for a repressor protein that can switch on/off the lac operon by binding to the operator. The difference between this and the repressor is that the repressor it is inactive by itself, but active when the something (the copressor) binds to it Here, the pathway is usually off- no B-galactosidase is made, except when there is an inducer, in this case allolactase, an isomer of lactose that is formed in small amounts of lactose. When this enzyme binds to the repressor, it inactivates it, thus starting the transcription process and allowing the B-galactosidase to be made. Both help the cell keep from wasting energy- one by not making an enzyme when there is nothing for it to break down, and one by not synthesizing amino acids when they are already present in the cell.

Positive Gene Regulation Activator- a protein that binds to DNA and stimulates transcription of a gene. Both of the previous example exhibit negative control of genes, because the operons are switched off by the negative form of the proteins. Gene regulation is positive when a regulatory protein interacts directly with the genome to switch transcription on. E.Coli use glucose over lactose, and enzymes necessary for glucose breakdown are constantly present. Only when glucose is in short supply, does it use lactose and generate the enzymes necessary for its breakdown. cAMP accumulates when glucose is scarce and binds to an inactivated CAP (a regulatory protein- called an activator), which binds to the promoter region, starting the synthesis of glucose. When glucose concentrations in the cell increase, cAMP falls, which results in CAP pulling away from the promoter and slowing the production of glucose. (Glucose is still synthesized, although at much lower levels because RNA polymerase is poorly bound to the DNA strand. When glucose is low (and lactose is present), cAMP Is high which activates cAMP. CAP then binds to DNA and causes snythesis of the enzymes needed to break down lactose. (B-galactosidase When glucose is present, e.coli doesn’t need to break down lactose. High glucose = low cAMP, so CAP isnt active to help in transcription. Transcription still happens just at low levels. The absence/presence of allolactose determines whether or not transcription happens at all. The amount of glucose (and the amount of cAMP) determines the rate of transcription if no repressor is present. Thus lac operon is under positive and negative gene control. (on/off switch and volume control) CAP is also known to be involved in the expression of 100 other genes

Exit Slip A certain mutation in E.coli changes the lac operator so that the active repressor cannot bind. How would this affect the cells production of B-galactosidase? The enzyme would continue to be made, which isn’t a problem, but is energetically inefficient

Warm Up Exercise Explain the difference in an inducer and a repressor operon.

Gene Expression Differential Gene Expression- the expression of different genes by cells with the same genome. A typical human cell might express about 20% of its protein-coding genes at any given time. Highly differentiated cells, such as muscles or nerves, express an even smaller percentage of their genes. Almost all cells contain an identical genome (immune cells are an exception- discussed later). The genes expressed in each cell type are unique. Each stage represents an opportunity for gene expression to be controlled. Note that prokaryotic cells do not have as many opportunities because they transcribe and translate simultaneously.