CHAPTER 14
• Gene regulation is the phenomenon in which the level of gene expression can vary
under different conditions. These genes are regulated so that the protein they
encode can be produced at the proper times and in the proper amounts. Therefore,
the cell avoids wasting valuable energy making proteins it does not need.
- Gene regulation is important because bacteria exist in an environment that is
frequently changing temperature, nutrients and many other factors.
In comparison, unregulated genes (= constitutive genes) have essentially constant levels of
expression in all condition over time. These genes encode for proteins that are continuously
needed for survival of the bacterium and are expressed in all cell types.
• A few common processes regulated at the genetic level:
- Metabolism
- Response to environmental stress
- Cell division
- Differentiation and development
- Several other reasons.
Gene regulation can occur at any of the steps in the pathway of gene expression
(transcription, translation & posttranslation).
Paragraph 1- overview of transcriptional regulation
• Transcriptional regulation involves the actions of regulatory proteins that can bind to
the DNA and affect the rate of transcription of one or more nearby genes. 2 types of
regulatory proteins are:
- Repressor > negative control
- Activator > positive control
Small effector molecules often play a critical role in transcription regulation. They are
sometimes called ligands.
- They do not bind directly to the DNA to alter transcription, but they exert their
effects by binding to a repressor or activator (regulatory protein). The binding of the
effector molecule causes a conformational change in the regulatory protein and
thereby influences whether the protein can bind to the DNA.
Genetic regulatory proteins that respond to small effector molecules typically have 2 binding
sites: 1 site is where the protein binds to the DNA, the other binding site is for the effector
molecule.
• Regulatory proteins are given names describing how they affect transcription when
they are bound to DNA, but small effector molecules are given names that describe
how they affect transcription when they are present in the cell at a sufficient
concentration to exert their effect.
- Inducer: causes the rate of transcription to increase. Genes that are regulated in this
manner are called inducible genes. Genes regulated in this manner= inducible.
An inducer may accomplish this in 2 ways: it can bind to a repressor protein and prevent it
from binding to the DNA, or it can bind to an activator protein and cause it to bind to DNA.
- Corepressor: bind to a repressor protein and thereby causing the protein to bind to
DNA.
- Inhibitor: binds to an activator protein and prevents it from binding DNA.
Both corepressor and inhibitors act to reduce the rate of transcription. The genes they
regulate are termed repressible genes.
,Paragraph 2- regulation of the lac operon
Enzymes are composed of proteins.
• Enzyme adaptation= A particular enzyme appears within a living cell only after the
cell has been exposed to the enzyme’s substrate. When a bacterium is not exposed
to a particular substance, it does not make the enzymes needed to metabolize that
substance.
• Jacob and Monod focused their attention of lactose metabolism in E. coli to
investigate in this phenomenon. This led to these understandings of regulation of Lac
operon:
- Exposure of bacterial cells to lactose increased the level of lactose utilizing enzymes
by 1000- to 10,000-fold.
- Antibody and labeling techniques revealed that the increase in the activity of these
enzymes was due to the increased synthesis of the proteins that form the enzymes.
- The removal of lactose caused an abrupt termination in the synthesis of enzymes
- The analysis in mutations in the lac operon revealed that each protein involved with
lactose utilization is encoded by a separate gene.
This concluded that enzyme adaptation is due to the synthesis of specific proteins in
response to lactose in the environment.
• In bacteria, it is common for a few genes to be arranged together in an operon (=a
regulatory unit consisting of a few structural genes under the control of one
promotor and one terminator.). An operon encodes a polycistronic (poly- ORF) mRNA
that contains the coding sequence for 2 or more structural proteins,
One biological advantage of an operon is that it allows a bacterium to coordinately regulate
a group of 2 or more genes that are involved in the same function: the expression of the
genes occurs as a single unit.
- To facilitate transcription, an operon is flanked by a promotor that signals the
beginning of transcription and a terminator that signals the end of transcription.
Promotor consists of operator, terminator and structural genes.
! bacterial genes are closely packed and can be transcribed in opposite directions.
• Operons involved in catabolism (= breakdown of a substance) are typically inducible.
> the substance to be broken down acts as the inducer.
• Operons involved in anabolism (= biosynthesis of a substance) are typically
repressible.
• 2 distinct transcriptional units are present in the organization of genes in E. coli in
lactose.
1. Lac operon: a CAP site, lac promotor, an operator site, three protein encoding genes:
lacZ, lacY, lacA, and a terminator.
- lacZ genes encodes beta galactosidase, enzymatically cleaves lactose and lactose
analogues. Also converts lactose into allolactose. Allolactose acts as a small effector
molecule for regulating the lac operon.
- The lacY gene encodes lactose permease, a membrane protein required for the active
transport of lactose into the cytoplasm of the bacterium.
- The lacA gene encodes galactoside transacetylase, an enzyme that covalently
modifies lactose and lactose analogs by the attachment of hydrophobic acetyl
groups. The acetylation of nonmetabolizable lactose analogs prevents their toxic
buildup within the bacterial cytoplasm by allowing them to diffuse out of the cell.
, • The CAP site and the operator site are short DNA segments that function in gene
regulation.
- The CAP site is recognized by an activator protein called catabolite activator protein
(CAP).
- The operator site is a sequence of bases that provides a binding site for a repressor
protein called lac repressor.
- The promotor is bound by RNA polymerase.
2. lacI gene: codes for a regulatory protein= repressor.
- The lacI gene has its own promotor called the i promotor and is constitutively
expressed at low levels.
- The lacI encodes lac repressor. This protein regulates the lac operon by binding to the
operator site and repressing transcription. This lac repressor is a homotetramer, a
protein composed of four identical subunits. Only a small amount of lac repressor is
needed to repress the lac operon. (There are usually 10 tetramers per cell)
The lac operon can be transcriptionally regulated in more than 1 way:
1. The first mechanism that is examined is inducible and under negative control. This
form of regulation involves lac repressor, which binds to the sequence of nucleotides
found within the lac operator site.
- Once bound, lac repressor prevents RNA polymerase from transcribing lacZ, lacY and
lacA genes.
The binding of the repressor to the operator site is reversible. In absence of allolactose, lac
repressor is bound to the operator site most of the time. Each of the repressor protein’s 4
subunits has a single binding site for allolactose, the inducer. When allolactose binds to
allosteric sites, a conformational change occurs that prevents lac repressor from binding to
the operator site. In this case, the RNA polymerase is free to transcribe the operon. > operon
has been induced.
- Allosteric regulation= action of a small effector molecule, such as allosteric proteins.
- Each subunit of a lac repressor has a region that binds to DNA and another region
that contains the allolactose binding site.
Lac operon mutants: lactose cannot be metabolized. Possible causes: base substitutions/
deletions/ insertions in the lac operon DNA.
- Constitutive expression (lacI-)= lac operon is always on even if there is no lactose>
may result in an inability to synthesize any repressor protein or it is unable to binds
to DNA.
- Super- repressor mutation (lacIs)= the lac operon cannot be induced even in the
presence of lactose and therefore remain bound to the operator.
- IQ = produces much more repressor than the normal I gene. Probably due to a
promotor up mutation. You may need more of the inducer to switch on the lac
operon.
In absence of lactose, no inducer (allolactose) is available to bind to lac repressor. In reality,
the repressor does not completely inhibit transcription, so very small amounts of beta
galactosidase, lactose permease, and galactoside transacetylase are made. The levels are for
too low to enable the bacterium to readily use lactose.
When the bacterium is exposed to lactose, a small amount can be transported into the
cytoplasm via lactose permease and beta galactosidase converts some of that lactose to
allolactose. The binding of allolactose promotes a conformation change that prevents the
, repressor from binding to the lac operator site. Translation of encoded polypeptides
produces proteins needed for lactose uptake and metabolism.
- The likelihood that allolactose will bind to the repressor depends on the allolactose
concentration.
• Jacob, Monod and pardee applied a genetic approach to understand the lacI-
mutation. Therefore, they involved conjugations between recipient cells, termed F-
cells, and donor cells, which were Hfr strains that transferred a portion of bacterial
chromosome. Later experiments involved the transfer of circular segments of DNA
known as F factors. They identified F’ factors that carried the lacI gene and the lac
operon. These F’ factors can be transferred from 1 cell to another by bacterial
conjugation.
- Merozygote= making haploid to diploid E coli. > 2 different lac operons.
The interactions between regulatory proteins and DNA sequences observed in this
experiment led to the definition of 2 genetic terms:
- trans- effect= form of genetic regulation that can occur even though 2 DNA segments
are not physically adjacent. The action of lac repressor on the lac operon is a trans
effect. A regulatory protein such as lac repressor is called trans- acting factors.
- Cis- acting element= DNA segment that must be adjacent to the genes that is
regulates and it has a cis- effect on gene expression.
Trans- effect is mediated by genes that encode regulatory proteins, whereas a cis- effect is
mediated by DNA sequences that are binding sites for regulatory proteins.
A mutation in a trans acting factor can be complemented by the introduction of a second
gene with normal function. However, a mutation in a cis acting element is not affected by
the introduction of a normal cis acting element into the cell.
2. The second way the lac operon is transcriptionally regulated, is known as catabolite
repression. This form of transcriptional regulation is influenced by the presence of
glucose, which is a catabolite (= a substance that is broken down inside the cell). The
presence of glucose ultimately leads to repression of the lac operon.
- When exposed to both lactose and glucose, E. Coli cells first use glucose, and
catabolite repression prevents the use of lactose. If glucose is used up, catabolite
repression is alleviated, and the bacterium then expresses the lac operon.
Diauxic growth= the sequential use of 2 sugars by a bacterium. Glucose, a more commonly
encountered sugar, is metabolized preferentially and then a second sugar is metabolized
after glucose Is depleted from the environment.
- Glucose is not itself the small effector molecule that binds directly to a genetic
regulatory protein. Instead, this from of regulation involves a small effector molecule,
cyclic- AMP (cAMP), which is produced from ATP via an enzyme known as adenylyl
cyclase.
cAMP binds an activator protein known as the CAP(Catabolite Activator Protein) or
CRP( cyclic AMP receptor protein). CAP is composed of 2 subunits, each of which binds one
molecule of cAMP.
- The cAMP- CAP complex is an example of regulation that is inducible and under
positive control.
• In the presence of glucose, the enzyme adenylyl cyclase is inhibited. This decreases
the levels of cAMP in the cell. Therefore, cAMP is no longer available to bind CAP.
The Lac transcription rate decreases.
• Gene regulation is the phenomenon in which the level of gene expression can vary
under different conditions. These genes are regulated so that the protein they
encode can be produced at the proper times and in the proper amounts. Therefore,
the cell avoids wasting valuable energy making proteins it does not need.
- Gene regulation is important because bacteria exist in an environment that is
frequently changing temperature, nutrients and many other factors.
In comparison, unregulated genes (= constitutive genes) have essentially constant levels of
expression in all condition over time. These genes encode for proteins that are continuously
needed for survival of the bacterium and are expressed in all cell types.
• A few common processes regulated at the genetic level:
- Metabolism
- Response to environmental stress
- Cell division
- Differentiation and development
- Several other reasons.
Gene regulation can occur at any of the steps in the pathway of gene expression
(transcription, translation & posttranslation).
Paragraph 1- overview of transcriptional regulation
• Transcriptional regulation involves the actions of regulatory proteins that can bind to
the DNA and affect the rate of transcription of one or more nearby genes. 2 types of
regulatory proteins are:
- Repressor > negative control
- Activator > positive control
Small effector molecules often play a critical role in transcription regulation. They are
sometimes called ligands.
- They do not bind directly to the DNA to alter transcription, but they exert their
effects by binding to a repressor or activator (regulatory protein). The binding of the
effector molecule causes a conformational change in the regulatory protein and
thereby influences whether the protein can bind to the DNA.
Genetic regulatory proteins that respond to small effector molecules typically have 2 binding
sites: 1 site is where the protein binds to the DNA, the other binding site is for the effector
molecule.
• Regulatory proteins are given names describing how they affect transcription when
they are bound to DNA, but small effector molecules are given names that describe
how they affect transcription when they are present in the cell at a sufficient
concentration to exert their effect.
- Inducer: causes the rate of transcription to increase. Genes that are regulated in this
manner are called inducible genes. Genes regulated in this manner= inducible.
An inducer may accomplish this in 2 ways: it can bind to a repressor protein and prevent it
from binding to the DNA, or it can bind to an activator protein and cause it to bind to DNA.
- Corepressor: bind to a repressor protein and thereby causing the protein to bind to
DNA.
- Inhibitor: binds to an activator protein and prevents it from binding DNA.
Both corepressor and inhibitors act to reduce the rate of transcription. The genes they
regulate are termed repressible genes.
,Paragraph 2- regulation of the lac operon
Enzymes are composed of proteins.
• Enzyme adaptation= A particular enzyme appears within a living cell only after the
cell has been exposed to the enzyme’s substrate. When a bacterium is not exposed
to a particular substance, it does not make the enzymes needed to metabolize that
substance.
• Jacob and Monod focused their attention of lactose metabolism in E. coli to
investigate in this phenomenon. This led to these understandings of regulation of Lac
operon:
- Exposure of bacterial cells to lactose increased the level of lactose utilizing enzymes
by 1000- to 10,000-fold.
- Antibody and labeling techniques revealed that the increase in the activity of these
enzymes was due to the increased synthesis of the proteins that form the enzymes.
- The removal of lactose caused an abrupt termination in the synthesis of enzymes
- The analysis in mutations in the lac operon revealed that each protein involved with
lactose utilization is encoded by a separate gene.
This concluded that enzyme adaptation is due to the synthesis of specific proteins in
response to lactose in the environment.
• In bacteria, it is common for a few genes to be arranged together in an operon (=a
regulatory unit consisting of a few structural genes under the control of one
promotor and one terminator.). An operon encodes a polycistronic (poly- ORF) mRNA
that contains the coding sequence for 2 or more structural proteins,
One biological advantage of an operon is that it allows a bacterium to coordinately regulate
a group of 2 or more genes that are involved in the same function: the expression of the
genes occurs as a single unit.
- To facilitate transcription, an operon is flanked by a promotor that signals the
beginning of transcription and a terminator that signals the end of transcription.
Promotor consists of operator, terminator and structural genes.
! bacterial genes are closely packed and can be transcribed in opposite directions.
• Operons involved in catabolism (= breakdown of a substance) are typically inducible.
> the substance to be broken down acts as the inducer.
• Operons involved in anabolism (= biosynthesis of a substance) are typically
repressible.
• 2 distinct transcriptional units are present in the organization of genes in E. coli in
lactose.
1. Lac operon: a CAP site, lac promotor, an operator site, three protein encoding genes:
lacZ, lacY, lacA, and a terminator.
- lacZ genes encodes beta galactosidase, enzymatically cleaves lactose and lactose
analogues. Also converts lactose into allolactose. Allolactose acts as a small effector
molecule for regulating the lac operon.
- The lacY gene encodes lactose permease, a membrane protein required for the active
transport of lactose into the cytoplasm of the bacterium.
- The lacA gene encodes galactoside transacetylase, an enzyme that covalently
modifies lactose and lactose analogs by the attachment of hydrophobic acetyl
groups. The acetylation of nonmetabolizable lactose analogs prevents their toxic
buildup within the bacterial cytoplasm by allowing them to diffuse out of the cell.
, • The CAP site and the operator site are short DNA segments that function in gene
regulation.
- The CAP site is recognized by an activator protein called catabolite activator protein
(CAP).
- The operator site is a sequence of bases that provides a binding site for a repressor
protein called lac repressor.
- The promotor is bound by RNA polymerase.
2. lacI gene: codes for a regulatory protein= repressor.
- The lacI gene has its own promotor called the i promotor and is constitutively
expressed at low levels.
- The lacI encodes lac repressor. This protein regulates the lac operon by binding to the
operator site and repressing transcription. This lac repressor is a homotetramer, a
protein composed of four identical subunits. Only a small amount of lac repressor is
needed to repress the lac operon. (There are usually 10 tetramers per cell)
The lac operon can be transcriptionally regulated in more than 1 way:
1. The first mechanism that is examined is inducible and under negative control. This
form of regulation involves lac repressor, which binds to the sequence of nucleotides
found within the lac operator site.
- Once bound, lac repressor prevents RNA polymerase from transcribing lacZ, lacY and
lacA genes.
The binding of the repressor to the operator site is reversible. In absence of allolactose, lac
repressor is bound to the operator site most of the time. Each of the repressor protein’s 4
subunits has a single binding site for allolactose, the inducer. When allolactose binds to
allosteric sites, a conformational change occurs that prevents lac repressor from binding to
the operator site. In this case, the RNA polymerase is free to transcribe the operon. > operon
has been induced.
- Allosteric regulation= action of a small effector molecule, such as allosteric proteins.
- Each subunit of a lac repressor has a region that binds to DNA and another region
that contains the allolactose binding site.
Lac operon mutants: lactose cannot be metabolized. Possible causes: base substitutions/
deletions/ insertions in the lac operon DNA.
- Constitutive expression (lacI-)= lac operon is always on even if there is no lactose>
may result in an inability to synthesize any repressor protein or it is unable to binds
to DNA.
- Super- repressor mutation (lacIs)= the lac operon cannot be induced even in the
presence of lactose and therefore remain bound to the operator.
- IQ = produces much more repressor than the normal I gene. Probably due to a
promotor up mutation. You may need more of the inducer to switch on the lac
operon.
In absence of lactose, no inducer (allolactose) is available to bind to lac repressor. In reality,
the repressor does not completely inhibit transcription, so very small amounts of beta
galactosidase, lactose permease, and galactoside transacetylase are made. The levels are for
too low to enable the bacterium to readily use lactose.
When the bacterium is exposed to lactose, a small amount can be transported into the
cytoplasm via lactose permease and beta galactosidase converts some of that lactose to
allolactose. The binding of allolactose promotes a conformation change that prevents the
, repressor from binding to the lac operator site. Translation of encoded polypeptides
produces proteins needed for lactose uptake and metabolism.
- The likelihood that allolactose will bind to the repressor depends on the allolactose
concentration.
• Jacob, Monod and pardee applied a genetic approach to understand the lacI-
mutation. Therefore, they involved conjugations between recipient cells, termed F-
cells, and donor cells, which were Hfr strains that transferred a portion of bacterial
chromosome. Later experiments involved the transfer of circular segments of DNA
known as F factors. They identified F’ factors that carried the lacI gene and the lac
operon. These F’ factors can be transferred from 1 cell to another by bacterial
conjugation.
- Merozygote= making haploid to diploid E coli. > 2 different lac operons.
The interactions between regulatory proteins and DNA sequences observed in this
experiment led to the definition of 2 genetic terms:
- trans- effect= form of genetic regulation that can occur even though 2 DNA segments
are not physically adjacent. The action of lac repressor on the lac operon is a trans
effect. A regulatory protein such as lac repressor is called trans- acting factors.
- Cis- acting element= DNA segment that must be adjacent to the genes that is
regulates and it has a cis- effect on gene expression.
Trans- effect is mediated by genes that encode regulatory proteins, whereas a cis- effect is
mediated by DNA sequences that are binding sites for regulatory proteins.
A mutation in a trans acting factor can be complemented by the introduction of a second
gene with normal function. However, a mutation in a cis acting element is not affected by
the introduction of a normal cis acting element into the cell.
2. The second way the lac operon is transcriptionally regulated, is known as catabolite
repression. This form of transcriptional regulation is influenced by the presence of
glucose, which is a catabolite (= a substance that is broken down inside the cell). The
presence of glucose ultimately leads to repression of the lac operon.
- When exposed to both lactose and glucose, E. Coli cells first use glucose, and
catabolite repression prevents the use of lactose. If glucose is used up, catabolite
repression is alleviated, and the bacterium then expresses the lac operon.
Diauxic growth= the sequential use of 2 sugars by a bacterium. Glucose, a more commonly
encountered sugar, is metabolized preferentially and then a second sugar is metabolized
after glucose Is depleted from the environment.
- Glucose is not itself the small effector molecule that binds directly to a genetic
regulatory protein. Instead, this from of regulation involves a small effector molecule,
cyclic- AMP (cAMP), which is produced from ATP via an enzyme known as adenylyl
cyclase.
cAMP binds an activator protein known as the CAP(Catabolite Activator Protein) or
CRP( cyclic AMP receptor protein). CAP is composed of 2 subunits, each of which binds one
molecule of cAMP.
- The cAMP- CAP complex is an example of regulation that is inducible and under
positive control.
• In the presence of glucose, the enzyme adenylyl cyclase is inhibited. This decreases
the levels of cAMP in the cell. Therefore, cAMP is no longer available to bind CAP.
The Lac transcription rate decreases.
