{"id":5,"date":"2019-08-16T17:55:08","date_gmt":"2019-08-16T21:55:08","guid":{"rendered":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/2019\/08\/16\/chapter-1\/"},"modified":"2022-06-10T20:26:19","modified_gmt":"2022-06-11T00:26:19","slug":"chapter-1","status":"publish","type":"chapter","link":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/chapter\/chapter-1\/","title":{"raw":"The lac operon","rendered":"The lac operon"},"content":{"raw":"<h2>What is an operon?<\/h2>\r\nIn prokaryote genomes, groups of [pb_glossary id=\"40\"]<strong>structural genes<\/strong>[\/pb_glossary] with related functions are often linked together, with their expression being controlled by a single set of\u00a0<strong>[pb_glossary id=\"38\"]regulatory elements[\/pb_glossary]<\/strong>.\u00a0These gene \"bundles\" are referred to as <strong>operons<\/strong>. Operons are an efficient way to streamline gene expression in prokaryotes. In this module we'll be looking specifically at the\u00a0<em>Escherichia coli<\/em>\u00a0<strong>lac operon<\/strong>, which is often used as a model system in genetics and has real, practical applications in molecular biology.\r\n<h3><span style=\"font-family: Roboto, Helvetica, Arial, sans-serif;font-size: 1.3rem;font-weight: bold\">1. The lac operon<\/span><\/h3>\r\n<em><strong>1.1 Structure<\/strong><\/em>\r\n\r\nThe lac operon contains three enzyme-coding structural genes\u00a0and three regulatory elements. The enzymes work together to allow\u00a0<em>E. coli<\/em> to digest the disaccharide <strong>[pb_glossary id=\"42\"]lactose[\/pb_glossary]<\/strong>, and the regulatory elements control the transcription of these enzymes.\r\n\r\nThese coding genes always come in a specific order within the operon, and during transcription, they are all transcribed together onto a single <strong>[pb_glossary id=\"60\"]polycistronic mRNA[\/pb_glossary]<\/strong> strand. Please explore Figure 1 thoroughly by clicking on the \"?\" icons, to familiarize yourself with the key regulatory elements, structural genes, and protein products of the lac operon.\r\n<div class=\"textbox textbox--examples\"><header class=\"textbox__header\">\r\n<p class=\"textbox__title\"><em><strong>1.2 Regulatory Elements<\/strong><\/em><\/p>\r\n\r\n<\/header>\r\n<div class=\"textbox__content\">\r\n<ul>\r\n \t<li><strong><em>Repressor<\/em> (<em>I<\/em>)<\/strong>: A coding sequence for the repressor protein. The repressor protein is a [pb_glossary id=\"61\"]<strong>trans-regulatory element<\/strong>[\/pb_glossary], and it's transcription is regulated by an entirely separate set of regulatory sequences.<\/li>\r\n \t<li><strong><em>Promoter<\/em> (<em>P<\/em>)<\/strong>: A non-coding [pb_glossary id=\"62\"]<strong>cis-regulatory element<\/strong>[\/pb_glossary]. <strong>[pb_glossary id=\"41\"]RNA polymerase[\/pb_glossary]<\/strong> (RNApol) must bind to the promoter region to begin mRNA transcription.<\/li>\r\n \t<li><strong><em>Operator<\/em> (<em>O<\/em>)<\/strong>: A non-coding cis-regulatory element. Contains a binding site for the repressor protein\u00a0<em>I<\/em>. When\u00a0<em>I<\/em> is bound to the operator, RNA polymerase cannot bind to the promoter.<\/li>\r\n<\/ul>\r\n<\/div>\r\n<\/div>\r\n<div class=\"textbox textbox--examples\"><header class=\"textbox__header\">\r\n<p class=\"textbox__title\"><em><strong>1.3 Structural Genes<\/strong><\/em><\/p>\r\n\r\n<\/header>\r\n<div class=\"textbox__content\">\r\n<ul>\r\n \t<li><strong><em>Beta-galactosidase<\/em> (<em>lac<\/em><em>Z<\/em>)<\/strong>: A coding sequence for beta-galactosidase, an enzyme that takes lactose as a substrate and cleaves it into the monosaccharides galactose and glucose. This is the first reactions necessary for the breakdown of lactose.<\/li>\r\n \t<li><strong><em>Permease<\/em> (<em>lac<\/em><em>Y<\/em>)<\/strong>: A coding sequence for permease, a membrane-bound protein that allows lactose to enter the cell.<\/li>\r\n \t<li><strong><em>Beta-galactoside transacetylase\u00a0(lacA)<\/em><\/strong>: A coding sequence for beta-galactoside transacetylase, an enzyme that adds acetyl groups to lactose and other galactose-containing sugars. The role of this enzyme in lactose digestion is not well defined, and we will mostly be leaving it out of our lac operon models.<\/li>\r\n<\/ul>\r\n<\/div>\r\n<\/div>\r\n<h4>Figure 1: The lac operon<\/h4>\r\nClick on the \"?\" icons in this Figure to see more information about the component parts of the operon.\r\n\r\n[h5p id=\"1\"]\r\n\r\n&nbsp;\r\n<h3>2. Function<\/h3>\r\nWe can see from Figure 1 that the lac operon coordinates the transcription of three enzymes with related functions. This is evidently very practical, but true beauty of this system lies in the fact that it ensures that these genes only get transcribed <em>under specific environmental conditions<\/em>.\r\n\r\nLactose is a relatively rare sugar, and\u00a0most\u00a0<em>E. coli\u00a0<\/em>don't need to be producing the beta-galactosidase and permease enzymes at a constant rate. Luckily for\u00a0<em>E. coli<\/em>, the lac operon\u00a0<strong>only activates in the presence of lactose<\/strong>! Watch this short video,\u00a0courtesy of Virtual Cell, to see how this is accomplished:\r\n\r\nIn order to understand this video, you'll need a good understanding of <strong>gene transcription<\/strong> and <strong>mRNA translation<\/strong>. If anything in this video seems unfamiliar, please take some time to brush up on these topics.\r\n\r\nhttps:\/\/youtu.be\/oBwtxdI1zvk\r\n\r\n<strong>Video Notes:<\/strong>\r\n<ul>\r\n \t<li>In this video, Virtual Cell never specifically refers to the operator and promoter regions, choosing instead to lump them into a single regulatory element called the \"Controlling region\". For this course, you'll need to consider them as separate elements within the operon.<\/li>\r\n \t<li>Remember, although it isn't explicitly referenced in this video,\u00a0<em>lacA<\/em> is always transcribed and translated along with\u00a0<em>lacZ\u00a0<\/em>and\u00a0<em>lacY<\/em>. The function of this gene product is still unclear, so it's left out of most educational resources.<\/li>\r\n<\/ul>\r\nHopefully, this video has given you a basic idea of how the lac operon functions. In Section 3, we'll take a deeper dive into how the individual components of the operon interact with each other by considering what happens if one or more of them is altered by a mutation.\r\n<h3>3. Mutations<\/h3>\r\nIn molecular biology, one of the most common methods for figuring out a gene's function is to mutate it and measure the resulting effects on its organism's phenotype. In this section, we'll be looking at a variety of mutations that can occur in lac operon genes, and discussing the effects of those mutations on\u00a0<em>E. coli<\/em>. To do this, we'll be using the following symbols to represent the individual components of the lac operon:\r\n<div class=\"textbox\"><em>I P O Z Y A<\/em><\/div>\r\nIn this model, all the genetic elements in the operon are lined up in the same orientation as they are in an actual\u00a0<em>E. coli\u00a0<\/em>genome (see Figure 1). Since the function of\u00a0<em>lacA\u00a0<\/em>is not yet well defined, we'll be leaving it out of this model more often than not. When all the sequences are\u00a0<strong>[pb_glossary id=\"43\"]wild type[\/pb_glossary]<\/strong>,\u00a0the lac operon functions normally. We'll represent this using the following notation:\r\n<div class=\"textbox\"><em>I<sup>+<\/sup> P<sup>+<\/sup> O<sup>+<\/sup> Z<sup>+<\/sup> Y<sup>+<\/sup> A<sup>+<\/sup><\/em><\/div>\r\nIf a given gene is mutated, we'll change the superscript above that gene. Listed below are the specific mutations we are going to be looking at for this course:\r\n<div class=\"textbox textbox--examples\">\r\n<div class=\"textbox__content\">\r\n<ul>\r\n \t<li><strong><em>Null mutation<\/em><\/strong>: Denoted by X<sup>-<\/sup> (where X can be any genetic element on the operon),\u00a0DNA sequences with this mutation have completely lost their normal activity. In protein-coding genes, this means no protein is produced. In regulatory genes, this means that regular binding sites are non-functional (ie. the RNApol binding site in the promoter region, and the RNApol binding site in the operator region).<\/li>\r\n \t<li><strong><em>Constitutive activity<\/em><\/strong>: Denoted by O<sup>c<\/sup>, this mutation is specific to the operator region. Constitutively active operator regions always block the binding of repressor protein to the operator region. This results in transcription of the operon whether or not lactose is present, because the repressor is unable to block RNApol from binding to the promoter.<\/li>\r\n \t<li><strong><em>Super-repressor<\/em><\/strong>: Denoted by I<sup>s<\/sup>, this mutation is specific to the repressor-coding gene. Super-repressor genes produce special repressor proteins, which can still bind to the operator but not to lactose.<\/li>\r\n<\/ul>\r\n<\/div>\r\n<\/div>\r\nIn these next exercises, we'll consider what happens in a typical haploid\u00a0<em>E. coli<\/em> when some of these mutations occur. As a hint, remember that all regulatory elements in the operon need to be functioning normally before any structural genes can be transcribed.\r\n\r\n[h5p id=\"2\"]\r\n\r\n[h5p id=\"3\"]\r\n\r\n[h5p id=\"5\"]\r\n\r\n[h5p id=\"11\"]\r\n\r\n[h5p id=\"12\"]\r\n\r\n[h5p id=\"13\"]\r\n\r\n&nbsp;\r\n<h3>4. Merodiploids<\/h3>\r\nTypically, we represent\u00a0<em>E. coli\u00a0<\/em>and other prokaryotes as being completely haploid, with only one circular chromosome and only one copy of each gene. You may remember, however, from our chapter on <strong>prokaryote genetics<\/strong> that this isn't always the case. Bacteria, including\u00a0<em>E. coli<\/em>, can acquire DNA from their environment (translation), from phages (transduction) or from other bacteria (conjugation). This may result in <em>E. coli<\/em> with two copies of certain genes! We call these partially diploid prokaryotes\u00a0<strong>merodiploids <\/strong>(\"mero-\" comes from the Greek word for \"part\", or \"partial\").\u00a0Merodiploids can be produced in a lab setting, using Hfr\/F+ strains of <em>E. coli<\/em>.\r\n\r\nMerodiploid\u00a0<em>E. coli<\/em> are a fantastic research tool. They allow us to examine how wild-type and mutated alleles interact within a living organism, with all the added bonuses of working with\u00a0<em>E. coli<\/em> (fast reproduction\/growth, easy colony maintenance, etc.) In this module, we'll be representing merodiploids using the following notation:\r\n<div class=\"textbox\"><em>I<sup>+<\/sup> P<sup>+<\/sup> O<sup>+<\/sup> Z<sup>+<\/sup> Y<sup>+<\/sup> A<\/em><sup><em>+<\/em>\u00a0<\/sup>\/ F'\u00a0<em>I<sup>+<\/sup> P<sup>+<\/sup> O<sup>+<\/sup> Z<sup>+<\/sup> Y<sup>+<\/sup> A<sup>+<\/sup><\/em><\/div>\r\nIn this notation, we show a chromosomal lac operon and an Hfr plasmid lac operon side by side. Again, we've included the\u00a0<em>lacA\u00a0<\/em>gene here for completeness, but will be leaving it out of our exercises.\r\n\r\nBecause merodiploids have two copies of a given set of genes, mutations affect them differently. For example, if a single copy of a protein coding gene is inactivated, the second copy may still continue to produce viable protein, effectively masking the mutation.\r\n\r\nTry out your understanding using this next set of exercises:\r\n\r\n[h5p id=\"6\"]\r\n\r\n[h5p id=\"7\"]\r\n\r\n[h5p id=\"14\"]\r\n\r\n&nbsp;\r\n<h3>5. Regulators and Effectors<\/h3>\r\nWe've seen in Section 2\u00a0that the lac operon has a built-in lactose sensor: the repressor protein. When there is no lactose present, the repressor\u00a0prevents lac operon products from being translated by binding to the operator region. When lactose is plentiful in the environment, it is taken up by the cell and binds to the repressor, removing its ability to bind to the operator region. In general, we call any molecule that modifies a protein's function in this way an <strong>effector molecule<\/strong>. To be a true effector, a molecule must modify a protein's activity by selectively binding at an [pb_glossary id=\"53\"]<strong>allosteric site<\/strong>[\/pb_glossary].\r\n\r\nIn molecular biology terms, we would say that the repressor protein is a <strong>negative regulator<\/strong> of the lac operon, because it's binding to the operon decreases transcription. In contrast, a <strong>positive regulator<\/strong> would be a molecule that binds to the operon and increases transcription. The lac operon does indeed have a positive regulator: <strong>Catabolite Activator Protein<\/strong>, or <strong>CAP<\/strong>. Keeping pace with the repressor protein,\u00a0CAP has its own effector molecule: cyclic AMP, or cAMP.\r\n\r\ncAMP is produced by\u00a0<em>E. coli<\/em> as a metabolic byproduct when glucose is scarce. It binds to the allosteric site on CAP, activating the protein and forming what we'll call the\u00a0<strong>cAMP-CAP complex<\/strong>. Thus activated, CAP binds to the lac operon promoter region, just upstream of the binding site for RNApol. This increases the affinity of the promoter region for RNApol, which leads to a huge increase in lac operon transcription (Figure 2). Without the cAMP-CAP complex, the lac operon is still transcribed in the presence of lactose, but at a much slower rate.\r\n<h4>Figure 2: The cAMP-CAP complex<\/h4>\r\n[h5p id=\"8\"]\r\n\r\nNow we might wonder, if the lac operon already has a negative regulator, why does it also need a positive regulator? Ultimately, it all comes down to efficiency. <em>E. coli\u00a0<\/em>are more efficient at digesting glucose than lactose, so when glucose is plentiful, it's wasteful to transcribe lac operon enzymes. The most efficient regulatory system would be one which activates not only in the presence of lactose, but also in the absence of glucose; this is what the cAMP-CAP complex accomplishes.\r\n\r\nTest your understanding using the next set of exercises:\r\n\r\n[h5p id=\"15\"]\r\n\r\n[h5p id=\"16\"]\r\n\r\n[h5p id=\"17\"]\r\n\r\n[h5p id=\"9\"]\r\n\r\n[h5p id=\"10\"]","rendered":"<h2>What is an operon?<\/h2>\n<p>In prokaryote genomes, groups of <a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_40\"><strong>structural genes<\/strong><\/a> with related functions are often linked together, with their expression being controlled by a single set of\u00a0<strong><a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_38\">regulatory elements<\/a><\/strong>.\u00a0These gene &#8220;bundles&#8221; are referred to as <strong>operons<\/strong>. Operons are an efficient way to streamline gene expression in prokaryotes. In this module we&#8217;ll be looking specifically at the\u00a0<em>Escherichia coli<\/em>\u00a0<strong>lac operon<\/strong>, which is often used as a model system in genetics and has real, practical applications in molecular biology.<\/p>\n<h3><span style=\"font-family: Roboto, Helvetica, Arial, sans-serif;font-size: 1.3rem;font-weight: bold\">1. The lac operon<\/span><\/h3>\n<p><em><strong>1.1 Structure<\/strong><\/em><\/p>\n<p>The lac operon contains three enzyme-coding structural genes\u00a0and three regulatory elements. The enzymes work together to allow\u00a0<em>E. coli<\/em> to digest the disaccharide <strong><a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_42\">lactose<\/a><\/strong>, and the regulatory elements control the transcription of these enzymes.<\/p>\n<p>These coding genes always come in a specific order within the operon, and during transcription, they are all transcribed together onto a single <strong><a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_60\">polycistronic mRNA<\/a><\/strong> strand. Please explore Figure 1 thoroughly by clicking on the &#8220;?&#8221; icons, to familiarize yourself with the key regulatory elements, structural genes, and protein products of the lac operon.<\/p>\n<div class=\"textbox textbox--examples\">\n<header class=\"textbox__header\">\n<p class=\"textbox__title\"><em><strong>1.2 Regulatory Elements<\/strong><\/em><\/p>\n<\/header>\n<div class=\"textbox__content\">\n<ul>\n<li><strong><em>Repressor<\/em> (<em>I<\/em>)<\/strong>: A coding sequence for the repressor protein. The repressor protein is a <a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_61\"><strong>trans-regulatory element<\/strong><\/a>, and it&#8217;s transcription is regulated by an entirely separate set of regulatory sequences.<\/li>\n<li><strong><em>Promoter<\/em> (<em>P<\/em>)<\/strong>: A non-coding <a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_62\"><strong>cis-regulatory element<\/strong><\/a>. <strong><a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_41\">RNA polymerase<\/a><\/strong> (RNApol) must bind to the promoter region to begin mRNA transcription.<\/li>\n<li><strong><em>Operator<\/em> (<em>O<\/em>)<\/strong>: A non-coding cis-regulatory element. Contains a binding site for the repressor protein\u00a0<em>I<\/em>. When\u00a0<em>I<\/em> is bound to the operator, RNA polymerase cannot bind to the promoter.<\/li>\n<\/ul>\n<\/div>\n<\/div>\n<div class=\"textbox textbox--examples\">\n<header class=\"textbox__header\">\n<p class=\"textbox__title\"><em><strong>1.3 Structural Genes<\/strong><\/em><\/p>\n<\/header>\n<div class=\"textbox__content\">\n<ul>\n<li><strong><em>Beta-galactosidase<\/em> (<em>lac<\/em><em>Z<\/em>)<\/strong>: A coding sequence for beta-galactosidase, an enzyme that takes lactose as a substrate and cleaves it into the monosaccharides galactose and glucose. This is the first reactions necessary for the breakdown of lactose.<\/li>\n<li><strong><em>Permease<\/em> (<em>lac<\/em><em>Y<\/em>)<\/strong>: A coding sequence for permease, a membrane-bound protein that allows lactose to enter the cell.<\/li>\n<li><strong><em>Beta-galactoside transacetylase\u00a0(lacA)<\/em><\/strong>: A coding sequence for beta-galactoside transacetylase, an enzyme that adds acetyl groups to lactose and other galactose-containing sugars. The role of this enzyme in lactose digestion is not well defined, and we will mostly be leaving it out of our lac operon models.<\/li>\n<\/ul>\n<\/div>\n<\/div>\n<h4>Figure 1: The lac operon<\/h4>\n<p>Click on the &#8220;?&#8221; icons in this Figure to see more information about the component parts of the operon.<\/p>\n<div id=\"h5p-1\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-1\" class=\"h5p-iframe\" data-content-id=\"1\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Figure 1\"><\/iframe><\/div>\n<\/div>\n<p>&nbsp;<\/p>\n<h3>2. Function<\/h3>\n<p>We can see from Figure 1 that the lac operon coordinates the transcription of three enzymes with related functions. This is evidently very practical, but true beauty of this system lies in the fact that it ensures that these genes only get transcribed <em>under specific environmental conditions<\/em>.<\/p>\n<p>Lactose is a relatively rare sugar, and\u00a0most\u00a0<em>E. coli\u00a0<\/em>don&#8217;t need to be producing the beta-galactosidase and permease enzymes at a constant rate. Luckily for\u00a0<em>E. coli<\/em>, the lac operon\u00a0<strong>only activates in the presence of lactose<\/strong>! Watch this short video,\u00a0courtesy of Virtual Cell, to see how this is accomplished:<\/p>\n<p>In order to understand this video, you&#8217;ll need a good understanding of <strong>gene transcription<\/strong> and <strong>mRNA translation<\/strong>. If anything in this video seems unfamiliar, please take some time to brush up on these topics.<\/p>\n<p><iframe loading=\"lazy\" id=\"oembed-1\" title=\"Lac Operon\" width=\"500\" height=\"375\" src=\"https:\/\/www.youtube.com\/embed\/oBwtxdI1zvk?feature=oembed&#38;rel=0\" frameborder=\"0\" allowfullscreen=\"allowfullscreen\"><\/iframe><\/p>\n<p><strong>Video Notes:<\/strong><\/p>\n<ul>\n<li>In this video, Virtual Cell never specifically refers to the operator and promoter regions, choosing instead to lump them into a single regulatory element called the &#8220;Controlling region&#8221;. For this course, you&#8217;ll need to consider them as separate elements within the operon.<\/li>\n<li>Remember, although it isn&#8217;t explicitly referenced in this video,\u00a0<em>lacA<\/em> is always transcribed and translated along with\u00a0<em>lacZ\u00a0<\/em>and\u00a0<em>lacY<\/em>. The function of this gene product is still unclear, so it&#8217;s left out of most educational resources.<\/li>\n<\/ul>\n<p>Hopefully, this video has given you a basic idea of how the lac operon functions. In Section 3, we&#8217;ll take a deeper dive into how the individual components of the operon interact with each other by considering what happens if one or more of them is altered by a mutation.<\/p>\n<h3>3. Mutations<\/h3>\n<p>In molecular biology, one of the most common methods for figuring out a gene&#8217;s function is to mutate it and measure the resulting effects on its organism&#8217;s phenotype. In this section, we&#8217;ll be looking at a variety of mutations that can occur in lac operon genes, and discussing the effects of those mutations on\u00a0<em>E. coli<\/em>. To do this, we&#8217;ll be using the following symbols to represent the individual components of the lac operon:<\/p>\n<div class=\"textbox\"><em>I P O Z Y A<\/em><\/div>\n<p>In this model, all the genetic elements in the operon are lined up in the same orientation as they are in an actual\u00a0<em>E. coli\u00a0<\/em>genome (see Figure 1). Since the function of\u00a0<em>lacA\u00a0<\/em>is not yet well defined, we&#8217;ll be leaving it out of this model more often than not. When all the sequences are\u00a0<strong><a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_43\">wild type<\/a><\/strong>,\u00a0the lac operon functions normally. We&#8217;ll represent this using the following notation:<\/p>\n<div class=\"textbox\"><em>I<sup>+<\/sup> P<sup>+<\/sup> O<sup>+<\/sup> Z<sup>+<\/sup> Y<sup>+<\/sup> A<sup>+<\/sup><\/em><\/div>\n<p>If a given gene is mutated, we&#8217;ll change the superscript above that gene. Listed below are the specific mutations we are going to be looking at for this course:<\/p>\n<div class=\"textbox textbox--examples\">\n<div class=\"textbox__content\">\n<ul>\n<li><strong><em>Null mutation<\/em><\/strong>: Denoted by X<sup>&#8211;<\/sup> (where X can be any genetic element on the operon),\u00a0DNA sequences with this mutation have completely lost their normal activity. In protein-coding genes, this means no protein is produced. In regulatory genes, this means that regular binding sites are non-functional (ie. the RNApol binding site in the promoter region, and the RNApol binding site in the operator region).<\/li>\n<li><strong><em>Constitutive activity<\/em><\/strong>: Denoted by O<sup>c<\/sup>, this mutation is specific to the operator region. Constitutively active operator regions always block the binding of repressor protein to the operator region. This results in transcription of the operon whether or not lactose is present, because the repressor is unable to block RNApol from binding to the promoter.<\/li>\n<li><strong><em>Super-repressor<\/em><\/strong>: Denoted by I<sup>s<\/sup>, this mutation is specific to the repressor-coding gene. Super-repressor genes produce special repressor proteins, which can still bind to the operator but not to lactose.<\/li>\n<\/ul>\n<\/div>\n<\/div>\n<p>In these next exercises, we&#8217;ll consider what happens in a typical haploid\u00a0<em>E. coli<\/em> when some of these mutations occur. As a hint, remember that all regulatory elements in the operon need to be functioning normally before any structural genes can be transcribed.<\/p>\n<div id=\"h5p-2\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-2\" class=\"h5p-iframe\" data-content-id=\"2\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 1\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-3\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-3\" class=\"h5p-iframe\" data-content-id=\"3\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 2\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-5\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-5\" class=\"h5p-iframe\" data-content-id=\"5\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 3\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-11\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-11\" class=\"h5p-iframe\" data-content-id=\"11\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 4\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-12\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-12\" class=\"h5p-iframe\" data-content-id=\"12\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 5\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-13\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-13\" class=\"h5p-iframe\" data-content-id=\"13\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 6\"><\/iframe><\/div>\n<\/div>\n<p>&nbsp;<\/p>\n<h3>4. Merodiploids<\/h3>\n<p>Typically, we represent\u00a0<em>E. coli\u00a0<\/em>and other prokaryotes as being completely haploid, with only one circular chromosome and only one copy of each gene. You may remember, however, from our chapter on <strong>prokaryote genetics<\/strong> that this isn&#8217;t always the case. Bacteria, including\u00a0<em>E. coli<\/em>, can acquire DNA from their environment (translation), from phages (transduction) or from other bacteria (conjugation). This may result in <em>E. coli<\/em> with two copies of certain genes! We call these partially diploid prokaryotes\u00a0<strong>merodiploids <\/strong>(&#8220;mero-&#8221; comes from the Greek word for &#8220;part&#8221;, or &#8220;partial&#8221;).\u00a0Merodiploids can be produced in a lab setting, using Hfr\/F+ strains of <em>E. coli<\/em>.<\/p>\n<p>Merodiploid\u00a0<em>E. coli<\/em> are a fantastic research tool. They allow us to examine how wild-type and mutated alleles interact within a living organism, with all the added bonuses of working with\u00a0<em>E. coli<\/em> (fast reproduction\/growth, easy colony maintenance, etc.) In this module, we&#8217;ll be representing merodiploids using the following notation:<\/p>\n<div class=\"textbox\"><em>I<sup>+<\/sup> P<sup>+<\/sup> O<sup>+<\/sup> Z<sup>+<\/sup> Y<sup>+<\/sup> A<\/em><sup><em>+<\/em>\u00a0<\/sup>\/ F&#8217;\u00a0<em>I<sup>+<\/sup> P<sup>+<\/sup> O<sup>+<\/sup> Z<sup>+<\/sup> Y<sup>+<\/sup> A<sup>+<\/sup><\/em><\/div>\n<p>In this notation, we show a chromosomal lac operon and an Hfr plasmid lac operon side by side. Again, we&#8217;ve included the\u00a0<em>lacA\u00a0<\/em>gene here for completeness, but will be leaving it out of our exercises.<\/p>\n<p>Because merodiploids have two copies of a given set of genes, mutations affect them differently. For example, if a single copy of a protein coding gene is inactivated, the second copy may still continue to produce viable protein, effectively masking the mutation.<\/p>\n<p>Try out your understanding using this next set of exercises:<\/p>\n<div id=\"h5p-6\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-6\" class=\"h5p-iframe\" data-content-id=\"6\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 7\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-7\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-7\" class=\"h5p-iframe\" data-content-id=\"7\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 8\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-14\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-14\" class=\"h5p-iframe\" data-content-id=\"14\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 9\"><\/iframe><\/div>\n<\/div>\n<p>&nbsp;<\/p>\n<h3>5. Regulators and Effectors<\/h3>\n<p>We&#8217;ve seen in Section 2\u00a0that the lac operon has a built-in lactose sensor: the repressor protein. When there is no lactose present, the repressor\u00a0prevents lac operon products from being translated by binding to the operator region. When lactose is plentiful in the environment, it is taken up by the cell and binds to the repressor, removing its ability to bind to the operator region. In general, we call any molecule that modifies a protein&#8217;s function in this way an <strong>effector molecule<\/strong>. To be a true effector, a molecule must modify a protein&#8217;s activity by selectively binding at an <a class=\"glossary-term\" aria-haspopup=\"dialog\" aria-describedby=\"definition\" href=\"#term_5_53\"><strong>allosteric site<\/strong><\/a>.<\/p>\n<p>In molecular biology terms, we would say that the repressor protein is a <strong>negative regulator<\/strong> of the lac operon, because it&#8217;s binding to the operon decreases transcription. In contrast, a <strong>positive regulator<\/strong> would be a molecule that binds to the operon and increases transcription. The lac operon does indeed have a positive regulator: <strong>Catabolite Activator Protein<\/strong>, or <strong>CAP<\/strong>. Keeping pace with the repressor protein,\u00a0CAP has its own effector molecule: cyclic AMP, or cAMP.<\/p>\n<p>cAMP is produced by\u00a0<em>E. coli<\/em> as a metabolic byproduct when glucose is scarce. It binds to the allosteric site on CAP, activating the protein and forming what we&#8217;ll call the\u00a0<strong>cAMP-CAP complex<\/strong>. Thus activated, CAP binds to the lac operon promoter region, just upstream of the binding site for RNApol. This increases the affinity of the promoter region for RNApol, which leads to a huge increase in lac operon transcription (Figure 2). Without the cAMP-CAP complex, the lac operon is still transcribed in the presence of lactose, but at a much slower rate.<\/p>\n<h4>Figure 2: The cAMP-CAP complex<\/h4>\n<div id=\"h5p-8\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-8\" class=\"h5p-iframe\" data-content-id=\"8\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Figure 2\"><\/iframe><\/div>\n<\/div>\n<p>Now we might wonder, if the lac operon already has a negative regulator, why does it also need a positive regulator? Ultimately, it all comes down to efficiency. <em>E. coli\u00a0<\/em>are more efficient at digesting glucose than lactose, so when glucose is plentiful, it&#8217;s wasteful to transcribe lac operon enzymes. The most efficient regulatory system would be one which activates not only in the presence of lactose, but also in the absence of glucose; this is what the cAMP-CAP complex accomplishes.<\/p>\n<p>Test your understanding using the next set of exercises:<\/p>\n<div id=\"h5p-15\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-15\" class=\"h5p-iframe\" data-content-id=\"15\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 10\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-16\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-16\" class=\"h5p-iframe\" data-content-id=\"16\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 11\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-17\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-17\" class=\"h5p-iframe\" data-content-id=\"17\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac Operon Exercise 12\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-9\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-9\" class=\"h5p-iframe\" data-content-id=\"9\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"Lac operon effectors\"><\/iframe><\/div>\n<\/div>\n<div id=\"h5p-10\">\n<div class=\"h5p-iframe-wrapper\"><iframe id=\"h5p-iframe-10\" class=\"h5p-iframe\" data-content-id=\"10\" style=\"height:1px\" src=\"about:blank\" frameBorder=\"0\" scrolling=\"no\" title=\"lac operon definitions\"><\/iframe><\/div>\n<\/div>\n<div class=\"glossary\"><span class=\"screen-reader-text\" id=\"definition\">definition<\/span><template id=\"term_5_40\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_40\"><div tabindex=\"-1\"><p>A structural gene codes for a product that does not regulate gene expression. Examples include enzymes, structural proteins, siRNA, etc.<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><template id=\"term_5_38\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_38\"><div tabindex=\"-1\"><p>Regulatory elements are non-coding regions of DNA that function to regulate gene expression. They may contain binding sites for polymerase enzymes, transcription factors, repressor proteins, etc.<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><template id=\"term_5_42\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_42\"><div tabindex=\"-1\"><p>A disaccharide made up of the two monosaccharides glucose and galactose.<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><template id=\"term_5_60\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_60\"><div tabindex=\"-1\"><p>A single mRNA strand that contains coding sequences for multiple products. Separate ribosome binding-sites exist for each coding sequence, allowing for simultaneous translation of all sequences.<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><template id=\"term_5_61\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_61\"><div tabindex=\"-1\"><p>DNA sequences that modify or regulate the expression of distant genes.<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><template id=\"term_5_62\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_62\"><div tabindex=\"-1\"><p>DNA sequence that modifies or controls the expression of an adjacent gene.<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><template id=\"term_5_41\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_41\"><div tabindex=\"-1\"><p>An enzyme that transcribes mRNA using DNA as a template<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><template id=\"term_5_43\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_43\"><div tabindex=\"-1\"><p>The most common form of a gene or phenotype found in nature<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><template id=\"term_5_53\"><div class=\"glossary__definition\" role=\"dialog\" data-id=\"term_5_53\"><div tabindex=\"-1\"><p>A binding site other than the protein's active site. In an enzyme, the active site is the site of catalysis. In a DNA-binding protein, the active site is the binding site for DNA.<\/p>\n<\/div><button><span aria-hidden=\"true\">&times;<\/span><span class=\"screen-reader-text\">Close definition<\/span><\/button><\/div><\/template><\/div>","protected":false},"author":792,"menu_order":1,"template":"","meta":{"pb_show_title":"","pb_short_title":"","pb_subtitle":"","pb_authors":[],"pb_section_license":""},"chapter-type":[47],"contributor":[],"license":[],"class_list":["post-5","chapter","type-chapter","status-publish","hentry","chapter-type-standard"],"part":3,"_links":{"self":[{"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/pressbooks\/v2\/chapters\/5","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/pressbooks\/v2\/chapters"}],"about":[{"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/wp\/v2\/types\/chapter"}],"author":[{"embeddable":true,"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/wp\/v2\/users\/792"}],"version-history":[{"count":25,"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/pressbooks\/v2\/chapters\/5\/revisions"}],"predecessor-version":[{"id":162,"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/pressbooks\/v2\/chapters\/5\/revisions\/162"}],"part":[{"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/pressbooks\/v2\/parts\/3"}],"metadata":[{"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/pressbooks\/v2\/chapters\/5\/metadata\/"}],"wp:attachment":[{"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/wp\/v2\/media?parent=5"}],"wp:term":[{"taxonomy":"chapter-type","embeddable":true,"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/pressbooks\/v2\/chapter-type?post=5"},{"taxonomy":"contributor","embeddable":true,"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/wp\/v2\/contributor?post=5"},{"taxonomy":"license","embeddable":true,"href":"https:\/\/pressbooks.bccampus.ca\/sfubisc202\/wp-json\/wp\/v2\/license?post=5"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}