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--- chapter_13 [2024/08/21 17:28] – [Questions and exercises] mike
+++ chapter_13 [2025/04/29 11:54] (current) – [Galactose metabolism in yeast] mike
@@ Line 1: / Line 1: @@
-<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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 $$ gal81 \cdot GAL4 \times gal4 \cdot GAL81 $$
-A surprising finding from this cross was that all the tetrads were of the parental ditype (PD); there were no tetratypes (TT) or nonparental ditypes (PD), indicating that $gal81$ and $gal4$ are very tightly linked(Another reminder to study tetrad analysis in [[Appendix_A|Appendix A]]!). Indeed, it turns out that $gal81$ maps to the coding region of the $gal4$ gene. In other words, $gal81$ is an allele of $gal4$; we say that "$gal81$ is allelic to $gal4$". The $gal81$ mutation was therefore renamed as $gal4^{81}$. Essentially $gal4^{81}$ behaves as a super-activator that is impervious to the negative effects of $gal80$. $gal4^{81}$ thus activates Gal1p expression independently of galactose and Gal80p. After cloning and sequencing $gal4^{81}$ and lots of other biochemical experiments, scientists learned that the $gal4^{81}$ mutation is a missense mutation that alters the amino acid sequence of the region of the Gal4p protein that normally interacts with Gal80p. This mutation prevents Gal80p from interacting with Gal4p. This was a very satisfying result, as genetics (a discipline concerned with abstract ways of thinking about biology) was used to learn  biochemical knowledge about how two gene products (proteins) physically interacted with each other to regulate gene expression.
+A surprising finding from this cross was that all the tetrads were of the parental ditype (PD); there were no tetratypes (TT) or nonparental ditypes (PD), indicating that $gal81$ and $gal4$ are very tightly linked((Another reminder to study tetrad analysis in [[Appendix_A|Appendix A]]!)). Indeed, it turns out that $gal81$ maps to the coding region of the $gal4$ gene. In other words, $gal81$ is an allele of $gal4$; we say that "$gal81$ is allelic to $gal4$". The $gal81$ mutation was therefore renamed as $gal4^{81}$. Essentially $gal4^{81}$ behaves as a super-activator that is impervious to the negative effects of $gal80$. $gal4^{81}$ thus activates Gal1p expression independently of galactose and Gal80p.
+After cloning and sequencing $gal4^{81}$ and lots of other biochemical experiments, scientists learned that the $gal4^{81}$ mutation is a missense mutation that alters the amino acid sequence of the region of the Gal4p protein that normally interacts with Gal80p. This mutation prevents Gal80p from interacting with Gal4p. This was a very satisfying result, as genetics (a discipline concerned with abstract ways of thinking about biology) was used to learn  biochemical knowledge about how two gene products (proteins) physically interact with each other to regulate gene expression.
@@ Line 127: / Line 131: @@
-These and many other genetic, molecular, and biochemical experiments led to the following model to explain Gal gene regulation. Upstream of the GAL1 gene (and other Gal genes), two cis-acting elements are needed for transcriptional activation. First, the TATA-binding protein (TBP) binds to the TATA consensus site  in the GAL1 promoter and provides 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 consensus site is also called the promoter; the promoter is usually around 40-50 bp of DNA in size. Note that the same word is used to describe cis-regulatory sequences in E. coli (Chap. 10); however, prokaryotic promoters and eukaryotic promoters are different from each other.
+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).
-RNAP binding to TBP alone does not enable transcription; the complex must be activated by a transcriptional activator (transactivator), which in this case is the Gal4 protein. The Gal4 protein binds to another cis-acting element in the Gal1 promoter region called the upstream activator sequence (UAS) that physically binds the Gal4 protein. The region of DNA that contains UAS is usually several hundred bp upstream from the promoter (see Chap. 14) and is commonly called an enhancer. In the absence of galactose, Gal80 physically prevents Gal4 from recruiting and activating RNAP. In the presence of galactose, the Gal80 protein changes conformation and binds to a different region of Gal4, unveiling the ability of Gal4 to recruit and activate RNAP. The mutation in the gal481 allele interferes with Gal80 binding, thereby allowing mutant Gal481 protein to recruit and activate RNAP all the time, even in the absence of galactose.
+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.
-Discussion Box: How does regulation of Gal genes in yeast compare to regulation of Lac operon genes in E. coli (seen in Chap. 10)? This is an important discussion to have!
+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 |}}
+<caption>
+Model of $GAL1$ regulation, based on genetic and biochemical analysis of Gal mutants. See text for details.
+</caption>
+</figure>
-Figure 13.5. Model of Gal1 regulation, based on genetic and biochemical analysis of Gal mutants.
+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.
-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.
-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!