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OpenStax-CNX module: m56876 1 Derived copy of Bis2A 14.1 Bacterial ∗ Gene Regulation Erin Easlon Based on Bis2A 14.1 Bacterial Gene Regulation† by OpenStax College Mitch Singer This work is produced by OpenStax-CNX and licensed under the Creative Commons Attribution License 4.0‡ Abstract By the end of this section, you will be able to: • Describe the steps involved in prokaryotic gene regulation • Explain the roles of activators, inducers, and repressors in gene regulation The DNA of bacteria and archaea is usually (there are a few known exceptions to the circular chromosome in bacteria) organized into a circular chromosome supercoiled in the nucleoid region of the cell cytoplasm. Proteins that are needed for a specic function, or that are involved in the same biochemical pathway, are often times encoded together in blocks called operons. Therefore, operons are single transcription units, encoding for multiple genes. Expression of these genes is organized from a single regulatory region and all genes in the operon are therefore regulated as a single unit. For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac ) operon. In bacteria, all transcription is controlled through RNA polymerase, a multiprotein complex that recognizes the promoter region and initiates transcription, elongates the transcript, and terminates transcription. Therefore, gene expression can be regulated at any of these steps, initiation, elongation, or termination; however, in bacteria, the majority of the regulation is at the level of transcription initiation. The Role of the Promoter The rst level of control of gene expression is at the promoter itself. There are two ways a promoter controls gene expression. First is which RNA polymerase holoenzyme (sigma + Core RNA polymerase) recognizes the promoter. Remember, bacteria have a number of sigma factors many of which control gene expression only under certain conditions, such as Sigma-S during stationary phase. The second level of control is promoter strenght, some promoters are considered "strong", while others are considered "weak". The basis of promoter strength is the specicity the promoter has to RNA polymerase. Each dierent sigma factor has a dierent recognition sequence, for example, the sigm-70 protein in E. coli has the recognition sequence 5'-TTGACA-(16-17 nucleotides)-TATAAT-3'. Strong promoters have sequences close to the consensus recognition sequence, weak promoters have sequences more divergent to the consensus. ∗ Version 1.1: Jul 17, 2015 2:51 pm -0500 † http://cnx.org/content/m56075/1.2/ ‡ http://creativecommons.org/licenses/by/4.0/ http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 2 Regulator Proteins The next layer of control is the addition of regulatory proteins. These proteins can either act to increase transcription, and are often called activators or activator proteins. These proteins bind to the promoter region and aid RNA polymerase to recognize a promoter and initiate transcription. Alternatively, regulatory proteins that inhibit transcription are often referred to as repressors or repressor proteins. Some regulatory proteins can act both as a repressor or an activator depending upon how they interact with RNA polymerase and the promoter. For example the regulatory protein called CAP can act to activate some genes and repress other genes. Therefore the terms "activator" and "repressor" should be used depending upon the situation or condition, and may not truely reect the role of the protein in question. Allosteric modulators of Protein Regulators Many regulatory proteins do not function independently, instead they rely on an allosteric regulatory molecule to control their activity. These small molecules are often referred to as inducers or co-repressors or coactivators. These small molecules are often metabolites, such as lactose or tryptophan or small regulatory molecules, such as cAMP or GTP. Below are some examples of regulatory systems that are controlled by repression and by activation. In some instances the presence of the small molecule activates or enhances the DNA-binding activity of the protein, thereby allowing the protein to bind better and regulate (either activate or repress) transcription initiation. Alternatively, the small molecule interacts with the regulatory protein and inhibits or decreases its ability to interact with the DNA or RNA polymerase, which can lead to either activation or repression of the gene it is controlling. 1 Repressors vs Activators: How do you tell Expression of the gene In general there are three states that can be used to described the expression of a gene or operon. The rst is constitutive. That the level of expression observed under most conditions. This level of expression could be very high, if the promoter is strong, or expression could be very little, if the promoter is weak. Regardless of the amount of expression, we observe very little change in expression under a variety of conditions. The second state is activation or induction, that is under a specic set of conditions, the expression of a gene or operon is increased or activated. Finally, expression of a gene can be decreased under a certain set of conditions, this is called repression. Mechanistically, in the last two cases, regulatory proteins are required to change the constitutive expression pattern. Whether a regulatory protein acts in a positive way, that is as an activator or acts in a negative way, as a repressor is not necessarily obvious. A simple test So how does one determine if a regulatory protein functions in a positive or negative way? A simple genetic test is to ask "what happens to expression if the regulatory protein is absent?" If a regulatory protein is acting positively, then its presence is required to activate gene expression. In its absence, there is no regulatory protein, therefore no activation, and the out come is no transcription. The phenotype of a null mutation in a regulatory protein is no activation. The opposite is true for a regulatory protein acting negatively. In its absence expression should be increased, because the gene keeping expression low is no longer around. Repression vs Activation What should become clear, is that how a regulatory protein functions: negatively or positively, may be independent as to what its eect is on gene expression. As you will see below, when E. coli is grown in the presence of lactose (and in the absence of glucose) expression of the lac operon is induced or activated. Yet, the protein regulator that is responsible for this expression phenotype is a repressor; it binds to the DNA to prevent transcription and a null mutation that removes the gene (lacI ) increases expression of the lac operon. In other words, the mechanism by which a regulatory protein work (positively or negatively) is independent as to how the gene or operon is expressed and behaves. This apparent contradiction can be rationalized when you incorporate the role of the allosteric regulator; in this case lactose. As you will see in the examples below, the key to the regulation is two fold: the mechanism by which the regulator works and the role and nature of the allosteric regulator. http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 2 The trp 3 Operon: A Repressor Operon Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded by ve genes. These ve genes are next to each other in what is called the tryptophan (trp ) operon (Figure 1). If tryptophan is present in the environment, then E. coli does not need to synthesize it and the switch controlling the activation of the genes in the trp operon is switched o. However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized. Figure 1: The ve genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed. A DNA sequence that codes for proteins is referred to as the coding region. The ve coding regions for the tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon. Just before the coding region is the transcriptional start site. This is the region of DNA to which RNA polymerase binds to initiate transcription. The promoter sequence is upstream of the transcriptional start site; each operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and regulate transcription. A DNA sequence called the operator sequence is encoded between the promoter region and the rst http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 4 coding gene. This operator contains the DNA code to which the repressor protein can bind. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to bind to the trp operator. Binding of the tryptophanrepressor complex at the operator physically prevents the RNA polymerase from binding, and transcribing the downstream genes. It should be noted that the term "operator" is limited to just a few systems and almost always refers to the binding site for a repressor. Conceptually what you need to remember is that there are sites on the DNA that interact with regulatory proteins allowing them to perform there appropriate function, repress transcription or activate transcription. These regulatory protein binding sites can vary as to location, but all control or regulate how RNA polymerase initiates transcription. When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned o, the trp operon is negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators. trp : http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 Watch this video1 to learn more about the 5 trp operon. 3 Catabolite Activator Protein (CAP): An Activator Regulator Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the operator sequences that act as a positive regulator to turn genes on and activate them. For example, when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate genes must be transcribed. When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. When glucose levels decline in the cell, accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that control the processing of alternative sugars. When cAMP binds to CAP, the complex binds to the promoter region of the genes that are needed to use the alternate sugar sources (Figure 2). In these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter. This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes. Please note, CAP-cAMP complex can also act as a repressor, depending upon where the binding site for CAP-cAMP is located. In the case of the Lactose operon, its position allows it to act as an activator; but in other operons it is positioned 3' or downstream from the promoter and can act as a repression. Figure 2: When glucose levels fall, E. coli may use other sugars for fuel but must transcribe new genes to do so. As glucose supplies become limited, cAMP levels increase. This cAMP binds to the CAP protein, a positive regulator that binds to an operator region upstream of the genes required to use other sugar sources. 1 http://openstaxcollege.org/l/trp_operon http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 4 The lac 6 Operon: An Inducer Operon The third type of gene regulation in prokaryotic cells occurs through inducible operons, which have proteins that bind to activate or repress transcription depending on the local environment and the needs of the cell. The lac operon is a typical inducible operon. As mentioned previously, E. coli is able to use other sugars as energy sources when glucose concentrations are low. To do so, the cAMPCAP protein complex serves as a positive regulator to induce transcription. One such sugar source is lactose. The lac operon encodes the genes necessary to acquire and process the lactose from the local environment. CAP binds to the operator sequence upstream of the promoter that initiates transcription of the lac operon. However, for the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed (Figure 3). This makes sense for the cell, because it would be energetically wasteful to create the proteins to process lactose if glucose was plentiful or lactose was not available. : http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 7 Figure 3: Transcription of the lac operon is carefully regulated so that its expression only occurs when glucose is limited and lactose is present to serve as an alternative fuel source. In E. coli, the case? trp operon is on by default, while the lac operon is o. Why do you think this is the If glucose is absent, then CAP can bind to the operator sequence to activate transcription. If lactose is absent, then the repressor binds to the operator to prevent transcription. If either of these requirements is met, then transcription remains o. Only when both conditions are satised is the lac operon transcribed (Table 1). http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 8 Signals that Induce or Repress Transcription of the lac Operon Glucose CAP binds Lactose Repressor binds Transcription + - - + No + - + - Some - + - + No - + + - Yes Table 1 : Watch an animated tutorial2 about the workings of 2 http://openstaxcollege.org/l/lac_operon http://cnx.org/content/m56876/1.1/ lac operon here. OpenStax-CNX module: m56876 9 5 Section Summary The regulation of gene expression in prokaryotic cells occurs at the transcriptional level. There are three ways to control the transcription of an operon: repressive control, activator control, and inducible control. Repressive control, typied by the trp operon, uses proteins bound to the operator sequence to physically prevent the binding of RNA polymerase and the activation of transcription. Therefore, if tryptophan is not needed, the repressor is bound to the operator and transcription remains o. Activator control, typied by the action of CAP, increases the binding ability of RNA polymerase to the promoter when CAP is bound. In this case, low levels of glucose result in the binding of cAMP to CAP. CAP then binds the promoter, which allows RNA polymerase to bind to the promoter better. In the last examplethe lac operontwo conditions must be met to initiate transcription. Glucose must not be present, and lactose must be available for the lac operon to be transcribed. If glucose is absent, CAP binds to the operator. If lactose is present, the repressor protein does not bind to its operator. Only when both conditions are met will RNA polymerase bind to the promoter to induce transcription. 6 Art Connections Exercise 1 Figure 3 In E. coli, the that this is the case? trp operon is on by default, while the (Solution on p. 10.) lac operon is o. Why do you think 7 Review Questions Exercise 2 If glucose is absent, but so is lactose, the a. b. c. d. lac operon will be ________. (Solution on p. 10.) activated repressed activated, but only partially mutated Exercise 3 (Solution on p. 10.) Bacteria and archaea lack a nucleus. Therefore, the genes in bacteria and archaea are: a. b. c. d. all expressed, all of the time transcribed and translated almost simultaneously transcriptionally controlled because translation begins before transcription ends b and c are both true 8 Free Response Exercise 4 (Solution on p. 10.) Exercise 5 (Solution on p. 10.) Exercise 6: Exercise 6 (Solution on p. 10.) Describe how transcription in bacteria can be altered by external stimulation such as excess lactose in the environment. What is the dierence between a repressible and an inducible operon? In the lac operon detailed above, LacI acts as a repressor and its allosteric regulator, lactose, acts as an inducer of the system. Redesign the lacI gene such that it no longer acts as a repressor but instead acts as an activator. What basic properties of the protein would need to change? http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 10 Solutions to Exercises in this Module to Exercise (p. 9) Figure 3 Tryptophan is an amino acid essential for making proteins, so the cell always needs to have some on hand. However, if plenty of tryptophan is present, it is wasteful to make more, and the expression of the trp receptor is repressed. Lactose, a sugar found in milk, is not always available. It makes no sense to make the enzymes necessary to digest an energy source that is not available, so the lac operon is only turned on when lactose is present. to Exercise (p. 9) B to Exercise (p. 9) D to Exercise (p. 9) Environmental stimuli can increase or induce transcription in bacteria. In this example, lactose in the environment will induce the transcription of the lac operon, but only if glucose is not available in the environment. to Exercise (p. 9) A repressible operon uses a protein bound to the promoter region of a gene to keep the gene repressed or silent. This repressor must be actively removed in order to transcribe the gene. An inducible operon is either activated or repressed depending on the needs of the cell and what is available in the local environment. Solution to Exercise (p. 9) First the Lac promoter would need to be much weaker promoter. Second, the LacI binding site (operator) would most likely need to move to the 5' end of the promoter. Third, when LacI binds to lactose, instead of decreasing the anity of the protein to the DNA, it would need to increase the binding anity. Similar to the way CAP-cAMP enhances the binding of CAP. In this hypothetical model, in the presence of lactose, lactose would bind LacI, cause a conrmation change that now stimulates LacI to bind to the promoter region and activate transcription. Hence, the expression pattern would look identical to as it does in the example above, except the mechanism of action by the regulator has changed from negative to positive. Glossary Denition 1: activator protein that binds to prokaryotic operators to increase transcription Denition 2: catabolite activator protein (CAP) protein that complexes with cAMP to bind to the promoter sequences of operons that control sugar processing when glucose is not available Denition 3: inducible operon operon that can be activated or repressed depending on cellular needs and the surrounding environment Denition 4: lac operon operon in prokaryotic cells that encodes genes required for processing and intake of lactose Denition 5: negative regulator protein that prevents transcription Denition 6: operator region of DNA outside of the promoter region that binds activators or repressors that control gene expression in prokaryotic cells Denition 7: operon collection of genes involved in a pathway that are transcribed together as a single mRNA in prokaryotic cells http://cnx.org/content/m56876/1.1/ OpenStax-CNX module: m56876 Denition 8: positive regulator protein that increases transcription Denition 9: repressor protein that binds to the operator of prokaryotic genes to prevent transcription Denition 10: transcriptional start site site at which transcription begins Denition 11: trp operon series of genes necessary to synthesize tryptophan in prokaryotic cells Denition 12: tryptophan amino acid that can be synthesized by prokaryotic cells when necessary http://cnx.org/content/m56876/1.1/ 11