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--- chapter_13 [2024/08/21 17:56] – [A general model for regulation of eukaryotic gene expression] mike
+++ chapter_13 [2025/04/29 11:54] (current) – [Galactose metabolism in yeast] mike
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-<typo fs:x-large>Chapter 13. Gene regulation in eukaryotes</typo>
+<-chapter_12|Chapter 12^table_of_contents|Table of Contents^chapter_14|Chapter 14->
+<typo fs:x-large>Chapter 13. %%Gene regulation in eukaryotes%%</typo>
 ===== Introduction =====
@@ Line 32: / Line 34: @@
-Once a gene (such as $gal1$) has been identified as being inducible under certain conditions (in this case by the addition of galactose), we can begin to dissect its regulatory mechanism by isolating mutants that defective in the regulatory process, i.e., mutants that constitutively express the $GAL$ genes even in the absence of galactose, and mutants that have lost the ability to induce the $GAL$ genes in the presence of galactose. If we were studying galactose regulation today, we would probably use a $lacZ$ reporter system similar to what we discussed in [[chapter_12|Chap. 12]].
+Once a gene (such as $gal1$) has been identified as being inducible under certain conditions (in this case by the addition of galactose), we can begin to dissect its regulatory mechanism by isolating mutants that are defective in the regulatory process, i.e., mutants that constitutively express the $GAL$ genes even in the absence of galactose, and mutants that have lost the ability to induce the $GAL$ genes in the presence of galactose. If we were studying galactose regulation today, we would probably use a $lacZ$ reporter system similar to what we discussed in [[chapter_12|Chap. 12]].
 <figure Fig2>
 {{ :gal_induction_mutants.png?400 |}}
 <caption>
-Using the mini-Tn7 strategy to find regulatory Gal mutants.
+Using the mini-Tn7 strategy to find regulatory Gal mutants. Glycerol is a carbon source for yeast that does not induce Gal genes.
 </caption>
 </figure>
@@ Line 74: / Line 76: @@
 </figure>
-$gal80$ mutant: The next useful regulatory mutant isolated was $gal80$, in which the $GAL1$-encoded galactokinase is expressed even in the absence of galactose and is not further induced in its presence. In other words, $gal80$ mutants are constitutive. Again, heterozygous diploids ($gal80$/$GAL80$) showed that $gal80$ is recessive, and mapping by tetrad analysis showed that $gal80$ is not linked to $gal1$, $gal4$ or any other $gal$ genes. If a mutant $gal80$ results in constitutive Gal1p expression, the simplest model to explain the data is that $GAL80$ negatively regulates Gal1p expression. Since $GAL4$ positively regulates and $GAL80$ negatively regulates Gal1p expression, we have to figure out how these two gene products work together to achieve such regulation. Assuming that $GAL4$ and $GAL80$ act in series (that is, in a linear genetic pathway), there are two formal possibilities:
+$gal80$ mutant: The next useful regulatory mutant isolated was $gal80$, in which the $GAL1$-encoded galactokinase is expressed even in the absence of galactose and is not further induced in its presence. In other words, $gal80$ mutants are constitutive. Again, heterozygous diploids ($\frac{gal80}{GAL80}$) showed that $gal80$ is recessive, and mapping by tetrad analysis showed that $gal80$ is not linked to $gal1$, $gal4$ or any other $gal$ genes. If a mutant $gal80$ results in constitutive Gal1p expression, the simplest model to explain the data is that $GAL80$ negatively regulates Gal1p expression. Since $GAL4$ positively regulates and $GAL80$ negatively regulates Gal1p expression, we have to figure out how these two gene products work together to achieve such regulation. Assuming that $GAL4$ and $GAL80$ act in series (that is, in a linear genetic pathway), there are two formal possibilities:
 <figure Fig4>
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 These and many other genetic, molecular, and biochemical experiments led to the following model (Figure {{ref>Fig5}}) to explain Gal gene regulation. Upstream of the $GAL1$ gene (and other Gal genes), two cis-acting DNA elements are needed for transcriptional activation: the promoter, and upstream activator sequences (UASs).
-First, TATA-binding protein (TBP) binds to a DNA sequence called the TATA consensus site (also called a TATA box), which is located just in front (upstream((In the context of gene expression, we use the terms upstream and downstream to describe directions on a gene. Relative to the transcription start site, any DNA positioned against the direction of transcription is described as upstream, and any DNA positioned with the direction of transcription is described as downstream.))) of the $GAL1$ transcription start site. TBP bound to the TATA box forms a scaffold for a very large RNA polymerase complex (RNAP). The area of DNA immediately upstream of the transcription start site of GAL1 that contains the TATA box is also called the promoter; the promoter is usually around 40-50 bp of DNA in size. Note that the word "promoter" is also used to describe cis-regulatory sequences in //E. coli// ([[chapter_10|Chap. 10]]); however, prokaryotic promoters and eukaryotic promoters do not work in the same way.
+First, TATA binding protein (TBP) binds to a DNA sequence called the TATA consensus site (also called a TATA box), which is located just in front (upstream((In the context of gene expression, we use the terms upstream and downstream to describe directions on a gene. Relative to the transcription start site, any DNA positioned against the direction of transcription is described as upstream, and any DNA positioned with the direction of transcription is described as downstream.))) of the $GAL1$ transcription start site. TBP bound to the TATA box forms a scaffold for a very large RNA polymerase complex. The area of DNA immediately upstream of the transcription start site of GAL1 that contains the TATA box is also called the promoter; the promoter is usually around 40-50 bp of DNA in size. Note that the word "promoter" is also used to describe cis-regulatory sequences in //E. coli// ([[chapter_10|Chap. 10]]); however, prokaryotic promoters and eukaryotic promoters do not work in the same way.
-RNAP binding to TBP alone does not enable transcription; the complex must be activated by a kind of protein called a transcriptional activator (or transactivator), which in this case is Gal4p. Gal4p binds to another DNA sequence further upstream of the $GAL1$ transcription start site called the upstream activator sequence (UAS). The region of DNA that contains UAS is usually several hundred bp upstream from the promoter (see [[chapter_14|Chap. 14]]) and is more generally called an enhancer. In the absence of galactose, Gal80p physically prevents Gal4p from activating RNAP. In the presence of galactose, the Gal80 protein changes conformation and binds to a different region of Gal4p, unveiling the ability of Gal4p to activate RNAP. The mutation in the $gal4^{81}$ allele interferes with Gal80p binding, thereby allowing mutant Gal4<sup>81</sup>p protein to recruit and activate RNAP all the time even in the absence of galactose.
+RNA polymerase binding to TBP alone does not enable transcription; the complex must be activated by a kind of protein called a transcriptional activator (or transactivator), which in this case is Gal4p. Gal4p binds to another DNA sequence further upstream of the $GAL1$ transcription start site called the upstream activator sequence (UAS). The region of DNA that contains UAS is usually several hundred bp upstream from the promoter (see [[chapter_14|Chap. 14]]) and is more generally called an enhancer. In the absence of galactose, Gal80p physically prevents Gal4p from activating RNAP. In the presence of galactose, the Gal80 protein changes conformation and binds to a different region of Gal4p, unveiling the ability of Gal4p to activate RNAP. The mutation in the $gal4^{81}$ allele interferes with Gal80p binding, thereby allowing mutant Gal4<sup>81</sup>p protein to recruit and activate RNAP all the time even in the absence of galactose.
 <figure Fig5>
+{{ :transcription_general_model.png?400 |}}
-Figure 13.5. Model of Gal1 regulation, based on genetic and biochemical analysis of Gal mutants.
+<caption>
-It is important to know that, while minor details will be different in different genes and organisms, the model shown in Fig. 13.5 generally holds true for all eukaryotic genes. This includes mammalian and human genes that may have biomedical or economic relevance.
+Model of $GAL1$ regulation, based on genetic and biochemical analysis of Gal mutants. See text for details.
+</caption>
 </figure>
+It is important to know that, while minor details will be different in different genes and organisms, the model shown in Fig. {{ref>Fig5}} generally holds true for all eukaryotic genes. This includes mammalian and human genes that may have biomedical or economic relevance.
-A final comment about the model for induction of the Gal genes by galactose: For many years it was assumed that galactose binds directly to the Gal80 protein, thus preventing it from inhibiting the Gal4 protein from activating Gal1 transcription. However, it now seems that one extra protein is involved in this chain of events. The Gal3 protein turns out to directly bind galactose. This allows Gal3 to move from the cytoplasm into the nucleus, upon which the galactose/Gal3 moiety binds to Gal80 to facilitate moving Gal80 to a different site on the Gal4 protein, thus allowing Gal4 to activate transcription. While the model as written in Fig. 13.5 does not include Gal3, the models are still formally correct.
+===== Closing thoughts =====
-In the next chapter we will be looking at structures of proteins and cis-regulatory elements, and how to genetically analyze them in more detail.
+A final comment about the model for induction of the Gal genes by galactose: For many years it was assumed that galactose binds directly to Gal80p, thus preventing it from inhibiting Gal4p from activating $GAL1$ transcription. However, one extra protein is involved in this chain of events. The Gal3 protein turns out to directly bind galactose. This allows Gal3p to move from the cytoplasm into the nucleus, upon which the galactose/Gal3p moiety binds to Gal80p to facilitate moving Gal80p to a different site on Gal4p, thus allowing Gal4p to activate transcription. While the model as written in Fig. {{ref>Fig5}} does not include Gal3p, the models are still formally correct.
+In the next chapter we will be looking at structures of some of these regulatory proteins and cis-regulatory elements, and how to genetically analyze them in more detail.
 ===== Questions and exercises =====
+Conceptual question: If you were one of the original scientists studying Gal gene regulation and you had a $gal4$ mutant, how could you clone $gal4$ by complementation? Your only tool you have for measuring phenotypes is measuring galactokinase activity (see footnote 2). Similarly, how could you clone $gal80$ by complementation?
-Exercise 13.1: If you were one of the original scientists studying Gal gene regulation, and you had a gal4 mutant, how could you clone gal4 by complementation?
-Exercise 13.2: Similarly, how could you clone gal80 by complementation?
+Exercise 1: In the chapter, we discuss that $gal81$ was shown to be allelic to $gal4$ by linkage, followed by cloning and sequencing. Could scientists have established that gal81 is allelic to gal4 using the complementation test? Why or why not?
-Exercise 13.3: How could you show that gal4 and gal80 are not alleles of the same gene?
+Exercise 2: $gal4^{81}$ and $gal80$ are both constitutive mutants (although one is dominant and one is recessive). Devise an experiment to show that $gal4^{81}$ and $gal80$ are not alleles of the same gene.
-Discussion Box: In the above paragraph, we discuss that gal81 was shown to be allelic to gal4 by linkage, followed by cloning and sequencing. Could scientists have established that gal81 is allelic to gal4 using the complementation test? Why or why not?
+Conceptual question: How does regulation of Gal genes in yeast compare to regulation of Lac operon genes in //E. coli// (seen in [[chapter_10|Chap. 10]])? This is an important thing to think about!