TNBGGA: The No Bullshit Guide to Genetic Analysis

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-<typo fs:x-large>Chapter 10. Gene regulation in %%bacteria%%</typo>+<-chapter_09|Chapter 09^table_of_contents|Table of Contents^chapter_11|Chapter 11-> 
 + 
 +<typo fs:x-large>Chapter 10. %%Gene regulation in bacteria%%</typo>
  
 We are now going to look at ways that genetics can be used to study gene regulation. Up to this point, we have examined gene function as something static and unchanging. But many prokaryotic and eukaryotic genes change their activity depending on the environment cells find themselves in. The question we wish to ask is: how do cells adjust the expression of genes in response to different environmental conditions?  We are now going to look at ways that genetics can be used to study gene regulation. Up to this point, we have examined gene function as something static and unchanging. But many prokaryotic and eukaryotic genes change their activity depending on the environment cells find themselves in. The question we wish to ask is: how do cells adjust the expression of genes in response to different environmental conditions? 
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 </figure> </figure>
  
-The LacY and LacZ proteins (formal names for the permease and β-galactosidase enzymes, respectively) are not expressed (produced) by the cell until lactose is present in the environment (Fig. {{ref>Fig1}}). Both the lacY and lacZ genes are part of a cluster of genes that are regulated together called an operon. The logic of the Lac operon is that the proteins required by the cell to use lactose as a food source are only made when the lactose is available. This prevents wasteful expression of proteins and enzymes when their substrates are not available.+The LacY and LacZ proteins (formal names for the permease and β-galactosidase enzymes, respectively) are not expressed (produced) by the cell until an inducer molecule such as lactose is present in the environment (Fig. {{ref>Fig1}}). Both the $lacY$ and $lacZ$ genes are part of a cluster of genes that are regulated together called an operon. The logic of the Lac operon is that the proteins required by the cell to use lactose as a food source are only made when the lactose is available. This prevents wasteful expression of proteins and enzymes when their substrates are not available. We say that the Lac genes are inducible. 
  
 At first, scientists noted that lactose is both an inducer and substrate for the enzymes of the Lac operon, and they incorrectly concluded that lactose was somehow acting as a template for the formation of the enzyme, which obviously makes no sense now, given our current knowledge. Later, compounds were discovered that could act as inducers but were not themselves substrates for the Lac enzymes. The classic example of such a “gratuitous inducer” is IPTG (isopropyl β-D-1-thiogalactopyranoside; Fig. {{ref>Fig2}}), which is an effective inducer of LacZ expression but isn’t hydrolyzed by β-galactosidase. The existence of compounds such as IPTG shows that recognition of the inducer is a separate molecular event from lactose breakdown. At first, scientists noted that lactose is both an inducer and substrate for the enzymes of the Lac operon, and they incorrectly concluded that lactose was somehow acting as a template for the formation of the enzyme, which obviously makes no sense now, given our current knowledge. Later, compounds were discovered that could act as inducers but were not themselves substrates for the Lac enzymes. The classic example of such a “gratuitous inducer” is IPTG (isopropyl β-D-1-thiogalactopyranoside; Fig. {{ref>Fig2}}), which is an effective inducer of LacZ expression but isn’t hydrolyzed by β-galactosidase. The existence of compounds such as IPTG shows that recognition of the inducer is a separate molecular event from lactose breakdown.
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 {{ :lac_operon_model.png?400 |}} {{ :lac_operon_model.png?400 |}}
  <caption>  <caption>
-Model of the Lac operon. Note that lacI is included in the diagram but it is not part of the Lac operon and is in fact located thousands of bp away from the other Lac genes. The diagram is not drawn to scale.+Model of the Lac operon. Note that $lacI$ is included in the diagram but it is not part of the Lac operon and is in fact located thousands of bp away from the other Lac genes. The diagram is not drawn to scale.
 </caption> </caption>
 </figure> </figure>
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 ^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^ ^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^
 ^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^  ^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^
-|  $lacI^+$ $lacZ^+$  |  No  |  Yes  |  This is the wild type (shown for comparison)  |+|  $lacI^+$ $lacZ^+$  |  No  |  Yes  |  This is the wildtype (shown for comparison)  |
 |  $lacI^-$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^-$ is a constitutive mutation  | |  $lacI^-$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^-$ is a constitutive mutation  |
 |  $lacI^-$ $lacZ^+$/$F'$ $lacI^+$ $lacZ^+$ (see note in Table legend)  |  No  |  Yes  |  $lacI^-$ is a recessive mutation  | |  $lacI^-$ $lacZ^+$/$F'$ $lacI^+$ $lacZ^+$ (see note in Table legend)  |  No  |  Yes  |  $lacI^-$ is a recessive mutation  |
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 ^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^ ^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^
 ^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^  ^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^
-|  $lacO^+$ $lacZ^+$  |  No  |  Yes  |  This is the wild type (shown for comparison)  |+|  $lacO^+$ $lacZ^+$  |  No  |  Yes  |  This is the wildtype (shown for comparison)  |
 |  $lacO^c$ $lacZ^+$  |  Yes  |  Yes  |  $lacO^c$ is a constitutive mutation  | |  $lacO^c$ $lacZ^+$  |  Yes  |  Yes  |  $lacO^c$ is a constitutive mutation  |
 |  $lacOc$ $lacZ^+$/$F’$ $lacO^+$ $lacZ^+$  |  Yes  |  Yes  |  $lacO^c$ is a dominant mutation  | |  $lacOc$ $lacZ^+$/$F’$ $lacO^+$ $lacZ^+$  |  Yes  |  Yes  |  $lacO^c$ is a dominant mutation  |
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 </table> </table>
    
-Based on other information, Jacob and Monod actually knew that $lacI$ and $lacO$ are different genes because they mapped to completely different locations on the //E. coli// chromosome (i.e., they are different genes based on a test of position; [[chapter_05|Chap. 5]]). In fact, $lacO$ mapped very close to $lacZ$, whereas $lacI$ is farther away from $lacZ$. However, if you didn't know their map positions, at a first glance the only difference between $lacO^c$ and $lacI^-$ is that $lacO^c$ is dominant and $lacI^-$ is recessive; therefore, you might think that on the basis of a dominance test we could tell whether we have a $lacO^c$ or a $lacI^–$ mutation. However, life is not so simple, because it is possible to find $lacI^–$ mutations that are dominant. Such mutations are known as $lacI^{-d}$. "-d" stands for dominant negative; dominant negative mutations are similar to recessive loss-of-function mutations in that they both have similar loss-of-function phenotypes, except one is dominant and one is recessive. Another name for dominant negative is antimorphic (see [[chapter_08|Chap. 8]]). +Based on other information, Jacob and Monod actually knew that $lacI$ and $lacO$ are different genes because they mapped to completely different locations on the //E. coli// chromosome (i.e., they are different genes based on a test of position; [[chapter_05|Chap. 05]]). In fact, $lacO$ mapped very close to $lacZ$, whereas $lacI$ is farther away from $lacZ$. However, if you didn't know their map positions, at a first glance the only difference between $lacO^c$ and $lacI^-$ is that $lacO^c$ is dominant and $lacI^-$ is recessive; therefore, you might think that on the basis of a dominance test we could tell whether we have a $lacO^c$ or a $lacI^–$ mutation. However, life is not so simple, because it is possible to find $lacI^–$ mutations that are dominant. Such mutations are known as $lacI^{-d}$. "$-d$" stands for dominant negative; dominant negative mutations are similar to recessive loss of function mutations in that they both have similar loss of function phenotypes, except one is dominant and one is recessive. Another name for dominant negative is antimorphic (see [[chapter_08|Chap. 8]]). 
  
 <table Tab3> <table Tab3>
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 ^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^ ^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^
 ^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^  ^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^
-^  $lacI^+$ $lacZ^+$  |  No  |  Yes  |  This is the wild type (shown for comparison)  |+^  $lacI^+$ $lacZ^+$  |  No  |  Yes  |  This is the wildtype (shown for comparison)  |
 |  $lacI^{-d}$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^{-d}$ is a constitutive mutation  | |  $lacI^{-d}$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^{-d}$ is a constitutive mutation  |
 |  $lacI^{-d}$ $lacZ^+$/$F’$ $lacI^+$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^{-d}$ is a dominant mutation  | |  $lacI^{-d}$ $lacZ^+$/$F’$ $lacI^+$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^{-d}$ is a dominant mutation  |
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-===== The cis/trans test (bleh) =====+===== The cis-trans test =====
  
 <table Tab4> <table Tab4>
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 Jacob and Monod found that the physical location of genes relative to each other affected their function. Unlike eukaryotes, //E. coli// only has a single chromosome. Therefore, all genes on the chromosome are on the same DNA molecule. Note in Table {{ref>Tab4}} that $lacI^{-d}$ is dominant no matter whether it is on the same piece of DNA as $lacZ^+$ (on the //E. coli// chromosome; another way to say this is "in cis") or a different piece of DNA as $lacZ^+$ (on the $F'$ plasmid; another way to say this is "in trans"). By contrast, $lacO^c$ is only dominant if it acts in cis; that is, $lacO^c$ only exhibits its dominant effect when it is on the same piece of DNA as $lacZ^+$. Jacob and Monod explained this by theorizing that the $lacI$ gene must produce a protein that is able to diffuse and attach to either chromosomal DNA or $F'$ plasmid DNA. They further hypothesized that $lacO$ (which they also knew was very closely linked to $lacZ$) represented not a protein-producing gene but rather a DNA sequence that controlled $lacZ$ expression - and it only worked if $lacO$ was physically linked to $lacZ$ on the same piece of DNA. Jacob and Monod found that the physical location of genes relative to each other affected their function. Unlike eukaryotes, //E. coli// only has a single chromosome. Therefore, all genes on the chromosome are on the same DNA molecule. Note in Table {{ref>Tab4}} that $lacI^{-d}$ is dominant no matter whether it is on the same piece of DNA as $lacZ^+$ (on the //E. coli// chromosome; another way to say this is "in cis") or a different piece of DNA as $lacZ^+$ (on the $F'$ plasmid; another way to say this is "in trans"). By contrast, $lacO^c$ is only dominant if it acts in cis; that is, $lacO^c$ only exhibits its dominant effect when it is on the same piece of DNA as $lacZ^+$. Jacob and Monod explained this by theorizing that the $lacI$ gene must produce a protein that is able to diffuse and attach to either chromosomal DNA or $F'$ plasmid DNA. They further hypothesized that $lacO$ (which they also knew was very closely linked to $lacZ$) represented not a protein-producing gene but rather a DNA sequence that controlled $lacZ$ expression - and it only worked if $lacO$ was physically linked to $lacZ$ on the same piece of DNA.
  
-The concept of cis- and trans-activating factors applies to eukaryotic gene regulation as well. In fact, proteins that bind to DNA and activate gene transcription (even in eukaryotes) are sometimes called transactivators. So far, we've seen that the Lac operon is negatively regulated; that is, the default state of the Lac operon is that it is expressed, but the $lacO$ repressor inactivates expression unless an inducer is present. In the next section, we will see an example of a transactivator in //E. coli// called MalT. +The concept of cis- and trans-acting factors applies to eukaryotic gene regulation as well. In fact, proteins that bind to DNA and activate gene transcription (even in eukaryotes) are sometimes called transactivators. So far, we've seen that the Lac operon is negatively regulated; that is, the default state of the Lac operon is that it is expressed, but the $lacO$ repressor inactivates expression unless an inducer is present. In the next section, we will see an example of a transactivator in //E. coli// called MalT. 
  
 To understand positive regulation, we first need a little more background information on uninducible mutations in the Lac operon. Until now we have been mostly considering mutations that lead to constitutive synthesis of β-galactosidase. It is also possible to get mutations that are uninducible(($lacZ^-$ mutants are also uninducible but that is trivial for the purpose of understanding gene regulation, since $lacZ$ codes for β-galactosidase.)). For example, a mutation in the promoter ($lacP^-$) is uninducible (Table {{REF>Tab5}}). $lacP^-$ promoter mutants in the Lac operon can be distinguished from simple $lacZ^–$ mutations since promoter mutations affect the $lacY$ and $lacA$ genes as well.  To understand positive regulation, we first need a little more background information on uninducible mutations in the Lac operon. Until now we have been mostly considering mutations that lead to constitutive synthesis of β-galactosidase. It is also possible to get mutations that are uninducible(($lacZ^-$ mutants are also uninducible but that is trivial for the purpose of understanding gene regulation, since $lacZ$ codes for β-galactosidase.)). For example, a mutation in the promoter ($lacP^-$) is uninducible (Table {{REF>Tab5}}). $lacP^-$ promoter mutants in the Lac operon can be distinguished from simple $lacZ^–$ mutations since promoter mutations affect the $lacY$ and $lacA$ genes as well. 
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 ^  Genotype  ^  MalQ expression?  ^^  Interpretation  ^ ^  Genotype  ^  MalQ expression?  ^^  Interpretation  ^
 ^  :::  ^  Without maltose  ^  With maltose  ^  :::  ^ ^  :::  ^  Without maltose  ^  With maltose  ^  :::  ^
-|  $malT^+$  |  No  |  Yes  |  This is the wild type (shown for comparison)  |+|  $malT^+$  |  No  |  Yes  |  This is the wildtype (shown for comparison)  |
 |  $malT^c$  |  Yes  |  Yes  |  $malT^c$ is constitutive  | |  $malT^c$  |  Yes  |  Yes  |  $malT^c$ is constitutive  |
 |  $malT^c$/$F'$ $malT^+$  |  Yes  |  Yes  |  $malT^-$ is a dominant mutation  | |  $malT^c$/$F'$ $malT^+$  |  Yes  |  Yes  |  $malT^-$ is a dominant mutation  |
chapter_10.1725229686.txt.gz · Last modified: 2024/09/01 15:28 by mike