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--- chapter_10 [2024/09/01 15:23] – mike
+++ chapter_10 [2024/09/01 23:33] (current) – mike
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-===== The cis/trans test (bleh) =====
+<-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?
+===== The Lac operon as a model for analysis of gene regulation =====
+The principles of gene regulation were first worked out by [[wp>François_Jacob|Francois Jacob]] and [[wp>Jacques_Monod|Jacques Monod]] in the 1960s-1970s studying the //E. coli// genes required for cells to use the sugar lactose as a nutrient.
+<figure Fig1>
+{{ :lac_growth_curve.png?400 |}}
+<caption>
+LacY and LacZ protein expression in //E. coli// is induced by the addition of an inducer molecule such as lactose.
+</caption>
+</figure>
+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.
+<figure Fig2>
+{{ :iptg_and_galactose.jpg?400 |}}
+<caption>
+The structure of IPTG (right), a potent inducer of the Lac operon, and galactose (left) for comparison. Although IPTG can induce the Lac operon, it is not a substrate for LacZ (β-galactosidase), whereas galactose is.
+</caption>
+</figure>
+The next major finding was the discovery of $lacI^–$ mutants. $lacI^–$ mutants are constitutive, meaning that they always express β-galactosidase at high levels regardless of whether there is an inducer present or not. $lacI^–$ mutants have apparently lost a component of the machinery the cell uses to turn off β-galactosidase expression.
+<figure Fig3>
+{{ :lac_operon_model.png?400 |}}
+ <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.
+</caption>
+</figure>
+We now tell you the story of the Lac operon backwards. We first tell you the conclusions that Jacob and Monod made from their experiments. The regulatory system turns out to be quite simple (Fig. {{ref>Fig3}}). Our goal is then to understand how the genetic experiments described below helped them reach those conclusions. The results are interesting and of general importance, but we are more interested in understanding their thinking and how they applied genetics to studying gene expression.
+The general model of the Lac operon is that the inducer (which can be either lactose or IPTG) has a net positive effect on Lac operon expression because the inducer is a negative regulator of the repressor LacI, which is itself a negative regulator of the gene for β-galactosidase. Some key details are described here:
+  * The $lacI$ gene codes for the Lac repressor protein, a DNA binding protein that binds to a DNA sequence called $lacO$. Lac repressor protein is always made, regardless of whether there is inducer present.
+  * RNA polymerase binds to a DNA sequence called $lacP$, also called the promoter. RNA polymerase binds to $lacP$, also regardless of whether there is inducer present.
+  * Lac repressor protein normally prevents RNA polymerase from transcribing the mRNA for the $lacZ$, $lacY$, and $lacA$ genes. It acts like a roadblock for RNA polymerase.
+  * When inducer (either lactose or IPTG) is present, it binds to Lac repressor protein and causes it to detach from the $lacO$ DNA. With the roadblock removed, RNA polymerase is then able to transcribe the mRNA for the Lac genes.
+We will now consider how regulatory mutants can be analyzed genetically. In essence, we want to know how Jacob and Monod figured out the mechanism described above. We will use as examples different mutations in the Lac system, but these genetic tests are very general and can be applied to most regulatory systems. For instance, in [[chapter_13|Chap. 13]] we will see how these concepts (but not the technical details) can be applied to studying gene regulation in yeast. The concepts are more important than the technical details!
+===== Testing for dominance in the Lac operon =====
+<table Tab1>
+<columns 100% *100%*>
+^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^
+^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^
+|  $lacI^+$ $lacZ^+$  |  No  |  Yes  |  This is the wildtype (shown for comparison)  |
+|  $lacI^-$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^-$ is a constitutive mutation  |
+|  $lacI^-$ $lacZ^+$/$F'$ $lacI^+$ $lacZ^+$ (see note in Table legend)  |  No  |  Yes  |  $lacI^-$ is a recessive mutation  |
+|  $lacI^+$ $lacZ^-$  |  No  |  No  |  $lacZ^-$ is uninducible  |
+|  $lacI^+$ $lacZ^-$/$F'$ $lacI^+$ $lacZ^+$  |  No  |  Yes  |  $lacZ^-$ is a recessive mutation  |
+|  $lacI^+$ $lacZ^-$/$F'$ $lacI^-$ $lacZ^+$  |  No  |  Yes  |  $lacI^-$ and $lacZ^-$ mutations complement each other; the mutations are in different genes  |
+</columns>
+<caption>
+Merodiploid analysis for dominance in the Lac operon. Note that $lacZ$ and $lacI$ are in different operons but fortuitously they are only about 3 kb apart; thus, they can easily exist on the same $F'$.
+</caption>
+</table>
+Using the dominance test in separate experiments, Jacob and Monod established that the $lacI^-$ and $lacZ^-$ mutations are recessive, which suggested that they are probably loss of function mutations (Table {{ref>Fig1}}). Also, since they are recessive, this further allows us to use the complementation test to determine whether the mutations are in two different genes. It turns out that they are indeed different genes, because $lacI$ and $lacZ$ complement each other. Jacob and Monod reasoned that since $lacI$ mutations cause a lactose-metabolizing enzyme (β -galactosidase) to always be produced, $lacI$ itself probably doesn't code for a lactose-metabolizing enzyme. Rather, its gene product probably regulates the function or production of β-galactosidase by "shutting it down". Conversely, since $lacZ$ mutants cannot produce β-galactosidase no matter what, $lacZ$ either positively regulates the production of β-galactosidase or codes for β-galactosidase itself (it winds up that the latter is true).
+A second type of constitutive mutant is known as a $lacO^c$ mutation. We later learn that this type of mutation inactivates the $lacO$ operator site. $lacO^c$ mutations are dominant as revealed in tests of the appropriate merodiploids:
+<table Tab2>
+<columns 100% *100%*>
+^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^
+^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^
+|  $lacO^+$ $lacZ^+$  |  No  |  Yes  |  This is the wildtype (shown for comparison)  |
+|  $lacO^c$ $lacZ^+$  |  Yes  |  Yes  |  $lacO^c$ is a constitutive mutation  |
+|  $lacOc$ $lacZ^+$/$F’$ $lacO^+$ $lacZ^+$  |  Yes  |  Yes  |  $lacO^c$ is a dominant mutation  |
+</columns>
+<caption>
+Tests for $lacO^c$ dominance using merodiploids.
+</caption>
+</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. 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>
+<columns 100% *100%*>
+^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^
+^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^
+^  $lacI^+$ $lacZ^+$  |  No  |  Yes  |  This is the wildtype (shown for comparison)  |
+|  $lacI^{-d}$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^{-d}$ is a constitutive mutation  |
+|  $lacI^{-d}$ $lacZ^+$/$F’$ $lacI^+$ $lacZ^+$  |  Yes  |  Yes  |  $lacI^{-d}$ is a dominant mutation  |
+</columns>
+</caption>
+Dominant negative alleles of $lacI$.
+</caption>
+</table>
+$lacO^c$ and $lacI^{-d}$ are both dominant, so we can't use the complementation test to determine if they are allelic to each other. We need a different test to help us distinguish between $lacO^c$ and $lacI^{-d}$. We will now consider a new genetic test that will let us distinguish $lacO^c$ operator constitutive from $lacI^{-d}$ repressor dominant negative mutations. This test is informative because it gives us further clues in understanding how the various Lac genes function.
+===== The cis-trans test =====
 <table Tab4>
@@ Line 19: / Line 122: @@
 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-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.
+<table Tab5>
+<columns 100% *100%*>
+^  Genotype  ^  β-galactosidase expression?  ^^  Interpretation  ^
+^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^
+|  $lacP^+$ $lacZ^+$  |  No  |  Yes  |  This is the wild type (shown for comparison)  |
+|  $lacP^-$ $lacZ^+$  |  No  |  No  |  $lacP^-$ is uninducible  |
+|  $lacP^-$ $lacZ^+$/$F’$ $lacP^+$ $lacZ^+$  |  No  |  Yes  |  $lacP^-$ is a recessive mutation  |
+|  $lacP^-$ $lacZ^+$/$F’$ $lacP^+$ $lacZ^-$ (see note below)  |  No  |  No  |  $lacP^-$ is cis-acting  |
+|  $lacP^-$ $lacZ^-$/$F’$ $lacP^+$ $lacZ^+$  |  No  |  Yes  |  :::  |
+</columns>
+<caption>
+Characterization of $lacP$ mutants. Note that this experiment also shows that $lacP$ and $lacZ$ do not complement each other and therefore must be mutations in the same gene. this fits with our definition of a gene as a DNA segment needed to make a protein, since the promoter is certainly needed for expression.
+</caption>
+</table>
+Another type of uninducible mutation is in $lacI$, where a Lac repressor protein is formed that binds to the $lacO$ operator DNA but does not bind inducer. These so-called "super repressor" mutations are designated as $lacI^s$ (Table {{ref>Tab6}}). Finding uninducible mutations in a regulatory gene such as $lacI$ suggests the possibility that there might be positive regulatory mechanisms for gene expression in other genes.
+<table Tab6>
+<columns 100% *100%*>
+^  Genotype  ^^  β-galactosidase expression?  ^  Interpretation  ^
+^  :::  ^  Without IPTG  ^  With IPTG  ^  :::  ^
+|  $lacI^+$ $lacZ^+$  |  No  |  Yes  |  This is the wild type (shown for comparison)  |
+|  $lacI^s$ $lacZ^+$  |  No  |  No  |  $lacI^s$ is uninducible  |
+|  $lacI^s$ $lacZ^+$/$F’$ $lacI^+$ $lacZ^+$  |  No  |  No  |  $lacI^s$ is a dominant mutation  |
+</columns>
+<caption>
+$lacI^s$ super repressor mutants.
+</caption>
+</table>
+===== Positive regulation in the Mal operon =====
+We will next consider how a different //E. coli// operon is regulated positively. The Mal operon encodes several genes necessary to take up and degrade maltose, a disaccharide composed of two glucose residues (Fig. {{ref>Fig4}}).
+<figure Fig4>
+{{ :maltose_pathway.png?400 |}}
+<caption>
+Maltose uptake metabolism in //E. coli//.
+</caption>
+</figure>
+Much like the Lac operon, the products of the Mal operon are induced when maltose is added to cells. Thus, maltose acts as an inducer (Fig. {{ref>Fig5}}).
+<figure Fig5>
+{{ :mal_growth_curve.png?400 |}}
+<caption>
+Expression of MalQ, a product of the //E. coli// Mal operon, is induced by addition of maltose.
+</caption>
+</figure>
+When mutants that affected the regulation of the Mal operon were isolated, the most common type consisted of uninducible mutations in a gene known as $malT$. We can apply dominance tests and cis/trans tests we just learned earlier in this chapter to $malT$ mutations. We obtain the following results:
+<table Tab7>
+<columns 100% *100%*>
+^  Genotype  ^  MalQ expression?  ^^  Interpretation  ^
+^  :::  ^  Without maltose  ^  With maltose  ^  :::  ^
+|  $malT^+$  |  No  |  Yes  |  maltose induces the Mal operon  |
+|  $malT^-$  |	No  |  No  |  $malT^-$ is uninducible  |
+|  $malT^-$/$F’$ $malT^+$ $malQ^+$  |  No  |  Yes  |  $malT^-$ is a recessive mutation  |
+|  $malT^-$ $malQ^+$/$F'$ $malT^+$ $malQ^-$  |  No  |  Yes  |  $malT^+$ is trans-acting (also see note below)  |
+|  $malT^-$ $malQ^-$/$F'$ $malT^+$ $malQ^+$  |  No  |  Yes  |  :::  |
+</columns>
+<caption>
+Genetic characterization of $malT$ mutants. Note: these lines also tells us that $malT^-$ and $malQ^-$ complement each other.
+</caption>
+</table>
+From Table {{ref>Tab7}}, it looks as if the $malT^-$ trait is not expressed either in cis or in trans (compare to $lacI^{-d}$). Because $malT^-$ is recessive, it makes more sense to consider the properties of the dominant $malT^+$ allele in the cis/trans test. Viewed in this way, the $malT^+$ trait is expressed in both cis and trans and therefore $malT$ is considered to be trans-acting.
+This behavior is different from any of the Lac mutations that we have discussed. The interpretation of this data is that $malT$ encodes a diffusible gene product (not a sequence/site on DNA) that is required for activation of transcription of the Mal operon. Unlike the LacI protein, which is a tetramer (a protein formed from 4 identical polypeptide subunits), the MalT protein functions as a monomer. Genes with functions like malT are usually called an activator (also called a transactivator). As shown in Fig. {{ref>Fig6}}, maltose binds to the MalT activator protein, which causes a conformational change in MalT and allows it to bind near to the promoter and to stimulate transcription. Note that the genes required for maltose uptake are located in an operon elsewhere on the chromosome, but these genes are also regulated by MalT.
+<figure Fig6>
+{{ :mal_operon_model.png?400 |}}
+<caption>
+Model of the Mal operon. It is very useful to compare this model with that of the Lac operon shown in Fig. {{ref>Fig3}}.
+</caption>
+</figure>
+In this model, a DNA site called the initiator is where the MalT activator protein binds, near the promoter to activate transcription. If you think about how mutations in an initiator site should behave in dominance and cis/trans tests, you will see why in practice it is difficult to distinguish initiator site mutations from promoter mutations (compare to $lacP$ and $lacO$ mutations, where there are genetic tests that can distinguish between them).
+It is also possible to isolate “super activator” $malT$ mutants that will bind to the initiator site and activate transcription regardless of whether the inducer maltose is present. Such alleles of the $malT$ gene are called $malT^c$ and their properties are given in Table {{ref>Tab8}}.
+<table Tab8>
+<columns 100% *100%*>
+^  Genotype  ^  MalQ expression?  ^^  Interpretation  ^
+^  :::  ^  Without maltose  ^  With maltose  ^  :::  ^
+|  $malT^+$  |  No  |  Yes  |  This is the wildtype (shown for comparison)  |
+|  $malT^c$  |  Yes  |  Yes  |  $malT^c$ is constitutive  |
+|  $malT^c$/$F'$ $malT^+$  |  Yes  |  Yes  |  $malT^-$ is a dominant mutation  |
+|  $malT^c$ $malQ^+$/$F'$ $malT^+$ $malQ^-$  |  Yes  |  Yes  |  $malT^c$ is trans-acting  |
+|  $malT^c$ $malQ^-$/$F'$ $malT^+$ $malQ^+$  |  Yes  |  Yes  |  :::  |
+</columns>
+<caption>
+Characterization of $malT^c$ mutants.
+</caption>
+</table>
+===== Questions and exercises =====
+Exercise 1: (Challenge question!) You isolate an //E. coli// mutant that is auxotrophic for raffinose, a trisaccharide comprised of galactose (a monosaccharide) and fructose (a disaccharide). You temporarily name this mutant $rafA$. In biochemical experiments you find that $rafA$ mutants do not express α-galactosidase, an enzyme required to metabolize raffinose, and that in wildtype //E. coli//, α-galactosidase expression is induced by raffinose. What experiments would you do to find out if $rafA$ codes for a metabolic enzyme vs a regulatory gene?\\
+Conceptual question: With regards to the Mal operon, it should also be possible to isolate dominant negative $malT$ mutants that will interfere with the function of the $malT^+$ wild type allele in a merodiploid. However, $malT^{-d}$ dominant negative mutants have not been isolated. By comparison, dominant negative $lacI$ mutants have been isolated (see Table {{ref>Tab4}}). Why do you think this is? How do you think dominant negative mutations work at the biochemical (protein) level? An important hint is that LacI functions as a tetrameric protein whereas MalT functions as a monomer. This is a very useful discussion to have because it reinforces the concept of the relationship between genes and genetic descriptions of mutations, and physical manifestations of phenotypes by way of the products of genes (i.e., proteins).