🫷 Week 9 - Operons

Transcription in Prokaryotes

Sigma Factor Function
 Sigma factor (σ) recognizes and binds to the -35 and -10 consensus sequences in the promoter region
 Positions RNA polymerase correctly to begin transcription

-10 Consensus Sequence
 AT-rich, making it prone to unwinding
 Facilitates the initiation of transcription

Transcription in Eukaryotes

Initiation
 Assembly of transcription factors (TFs) and RNA polymerase II causes 11–15 bp of surrounding DNA to unwind
 Template strand is positioned within the active site of RNA polymerase II

Promoter Structure
 Each gene has a unique regulatory promoter
 Contains distinct regulatory elements and unique cofactors to influence transcription
 Regulatory promoter works with the core promoter to initiate transcription

Section 16.2 Outline: Gene Regulation

Topics Covered
 1 Constitutive vs Inducible vs Repressible
 2 Regulation Overview
 3 Negative Inducible Operon
 4 Negative Repressible Operon
 5 lac Operon
 6 Mutations
 7 Positive Control and Catabolite Repression

Notes
 Operator or promoter mutations and the trp operon will not be covered
 Sections 16.3 and 16.4 will not be covered

Constitutive vs Inducible vs Repressible Genes

Constitutively Expressed Genes
 Certain gene products, such as tRNAs, rRNAs, ribosomal proteins, RNA polymerase subunits, and enzymes for housekeeping functions, are essential in almost all cells
 Genes for these products are continuously expressed in most cells (constitutive)

Inducible and Repressible Genes
 Some gene products are needed only under specific environmental conditions
 Inducible genes are expressed only when needed
 Repressible genes are shut down when their products are no longer needed

Regulation of Gene Expression

Regulation of Gene Expression
 Gene expression can be controlled at multiple levels
 Focus here is on regulation at the level of transcription initiation
 Requires binding of proteins, called transcription factors, to the DNA

DNA Binding Proteins

DNA binding proteins can act as activators or repressors of transcription
Common in both prokaryotes and eukaryotes
Contain domains of 60–90 amino acids that recognize specific DNA sequences and interact with DNA grooves
Motifs bind non-covalently to the promoter of a gene or operon
Can recruit RNA polymerase or inhibit its binding

Transcription in Bacteria: Operons

Genes a, b, c are transcribed as a single mRNA
Transcription is regulated by a regulator protein, which is a transcription factor
Regulator binds to the operator part of the promoter

Operon Structure
 An operon is a group of structural genes plus sequences that control transcription
 A separate regulator gene with its own promoter encodes a regulator protein that may bind the operator site to regulate mRNA transcription

Function
 mRNA is translated into proteins or enzymes
 The products of mRNA catalyze reactions in a biochemical pathway

Control Elements in Bacterial Operons

Operator
 Part of the operon that helps determine whether transcription can take place
 Overlaps with the 3' end of the promoter and the 5' end of the transcription start site of the first structural gene
 Operator sequence is unique for each operon

Regulation of Transcription in Operons

Promoter/Operator Function
 Regulates whether transcription of the operon occurs
 Sometimes transcription needs to be turned on, other times it needs to be shut down

Regulatory Proteins
 Negative regulatory proteins (repressors) inhibit transcription
 Positive regulatory proteins (activators) stimulate transcription

Operon Types
 Inducible operons are normally off and are turned on when needed
 Repressible operons are normally on and are turned off when needed
 Several varieties of control exist depending on individual cell needs

Negative Inducible Operon

General Function
 Transcription is normally off and must be turned on

Regulator Protein
 Regulator gene encodes an active repressor protein
 Repressor binds to the operator and blocks RNA polymerase from binding to the promoter
 Keeps transcription off
 Repressor must be inactivated or removed for transcription to proceed

Negative Inducible Operon: Role of Inducer

Repressor Activity
 Repressor is active in the absence of a co-factor
 Binds to the operator and prevents transcription of structural genes

Inducer Function
 A small molecule called an inducer binds to the repressor and inactivates it
 Repressor can no longer bind to DNA
 RNA polymerase can activate transcription
 When the inducer is present, it binds to the regulator, making the regulator unable to bind to the operator
 Transcription takes place

General Notes
 Negative means the regulator protein is an inhibitor or repressor
 Inducible means something inactivates the repressor, which then induces transcription
 Usually involved in the degradation or metabolism of molecules

Negative Inducible Operon: Stepwise Mechanism

Without Inducer
 Repressor protein binds to the operator region of the operon
 Prevents transcription of the structural genes
 Blocks RNA polymerase from accessing the promoter

With Inducer
 Inducer binds to the repressor protein, causing conformational changes
 Repressor can no longer bind to the operator
 RNA polymerase can access the promoter and initiate transcription of the structural genes
 Transcription takes place

Function
 This mechanism ensures that the genes are only transcribed when the inducer is present
 Often signals that the cell needs the products of those genes

Operon Components
 Promoter
 Operator
 Structural genes

Image with Repressor

Image with Inducer

Example: lac Operon (Negative Inducible Operon)

Lactose is the inducer in the lac operon of Escherichia coli
In the absence of lactose, the repressor binds to the operator, blocking transcription
When lactose is present, it binds to the repressor and inactivates it
Repressor can no longer bind to the operator
Genes responsible for lactose metabolism are transcribed

Negative Repressible Operon

General Function
 Transcription normally takes place and must be turned off

Regulator Protein
 Regulator is a repressor, similar to negative inducible operons
 Repressor is inactive in the absence of a co-factor
 Unable to bind to the operator or promoter/operator complex

Transcription
 Structural genes are transcribed until the repressor is activated by a co-factor
 Inactive repressor allows transcription to proceed

Negative Repressible Operon: Role of Corepressor

Corepressor Function
 Small molecule called a corepressor (co-factor) binds to the repressor
 Repressor/corepressor complex can now bind to the operator
 Binding inhibits transcription

Function
 Usually involved in biosynthesis necessary for the cell
 Genes are only turned on when needed and off when not needed, making expression efficient
 Negative means the regulator protein is an inhibitor or repressor
 Repressible means something activates the repressor, which then represses transcription

Example: trp Operon
 In Escherichia coli, product (e.g. tryptophan) builds up
 Product binds to the regulator protein, making it active
 Active repressor binds to the operator, preventing transcription

Example: trp Operon in E. coli

Repressor Protein
 In its inactive form, the repressor cannot bind to the operator
 Transcription of genes for tryptophan synthesis takes place

Co-repressor (Tryptophan)
 When tryptophan levels are high, tryptophan acts as a co-repressor
 Binds to the repressor protein

Transcription Inhibition
 Binding of tryptophan to the repressor causes a conformational change
 Active repressor binds to the operator and blocks transcription

Function
 Ensures genes for tryptophan synthesis are transcribed only when tryptophan levels are low
 Prevents the cell from wasting energy producing tryptophan when it is abundant

Negative Inducible Operon: Summary

Repressor Function
 Repressor binds to the operator and inhibits transcription

Inducer Role
 Inducer (L) binds to the repressor (R)
 Binding prevents the repressor from attaching to the operator
 Transcription continues

Mechanism
 Repressor + operator = transcription inhibited
 Repressor + inducer = repressor cannot bind operator, transcription proceeds

Negative Repressible Operon: Summary

Repressor Activity
 Repressor is inactive and cannot bind to the operator in the absence of a co-repressor

Co-repressor Role
 When co-repressor binds to the repressor, the repressor becomes active
 Active repressor binds to the operator and inhibits transcription

Mechanism
 Inactive repressor alone = transcription proceeds
 Repressor + co-repressor = transcription inhibited

Negative Inducible vs Negative Repressible Operons

Negative Inducible Operon
 Repressor is active by default and binds to the operator
 Blocks transcription of structural genes
 Inducer binds to the repressor, inactivating it
 Repressor can no longer bind the operator
 Transcription occurs
 Typically involved in degradation or metabolism of molecules

Negative Repressible Operon
 Repressor is inactive by default and cannot bind the operator
 Transcription of structural genes normally takes place
 Co-repressor binds to the repressor, activating it
 Active repressor binds to the operator
 Transcription is inhibited
 Typically involved in biosynthesis of molecules

Positive Control of Transcription

Activator Proteins
 Regulatory protein acts as an activator
 Activator binds to the operator or upstream of the operator to induce transcription

Catabolite Activator Protein (CAP)
 CAP is a positive activator of transcription
 Binds just upstream of the promoter
 Enhances binding of RNA polymerase to the promoter

Lac Operon Regulation

Negative Inducible Control
 Regulator protein is an inhibitor
 Allolactose acts as an inducer by inactivating the inhibitor

Positive Control
 CAP combined with cAMP enhances transcription

Lac Operon Enzymes and Function

Lactose Metabolism
 Lactose, found in milk, can be metabolized by E. coli
 Needs to be transported into the cell by permease (lacY)
 β-galactosidase (lacZ) breaks lactose into glucose and galactose
 β-galactosidase also converts lactose into allolactose
 Thiogalactoside transacetylase (lacA) is the third enzyme, function unclear

Operon Structure
 All enzymes are encoded by adjacent structural genes
 They share a common promoter (lacP)

Function Summary
 Permease actively transports lactose across the cell membrane
 β-galactosidase breaks lactose into galactose and glucose
 β-galactosidase also converts some lactose into allolactose, which acts as the inducer

Lac Operon Induction

Absence of Lactose
 Very little transcription of the operon occurs, but not none
 Repressor protein (lacI) binds to the operator (lacO) and inhibits transcription

Presence of Lactose
 Addition of lactose to the medium instead of glucose increases transcription of the lac operon about 1000X within 2–3 minutes
 Lactose acts as the inducer

Coordinate Induction
 Simultaneous synthesis of several proteins by a specific inducer molecule

Lac Operon: Mechanism When Lactose is Present

Allolactose Formation
 Lactose is converted into glucose, galactose, and some allolactose
 Allolactose keeps the operon in the “on” position

Repressor Inactivation
 Allolactose binds to the repressor, causing it to release from the operator
 Repressor cannot bind to the operator

Transcription and Translation
 RNA polymerase binds to the promoter and initiates transcription of lacZ, lacY, and lacA
 Structural genes are transcribed and translated

Termination
 Once lactose is depleted, no more allolactose is produced
 Repressor binds again to the operator, inhibiting transcription

Lac Operon: Initial Activation

Low-Level Transcription
 Repression by the repressor does not completely shut down transcription
 Low levels of transcription occur even when the repressor is bound

Low Enzyme Levels
 Low levels of permease (lacY) and β-galactosidase (lacZ) are always present in cells
 These basal amounts allow lactose to enter the cell and be converted into allolactose to inactivate the repressor

Lac Operon in Escherichia coli

Repressor Protein
 In the absence of lactose, the repressor binds to the operator
 Prevents transcription of genes responsible for lactose metabolism

Inducer Molecule (Allolactose)
 When lactose is present, it is converted into allolactose
 Allolactose binds to the repressor and causes a conformational change

Transcription Initiation
 Repressor can no longer bind to the operator
 RNA polymerase accesses the promoter and transcribes the genes needed for lactose metabolism

Function
 Ensures genes are transcribed only when lactose is available
 Allows the cell to efficiently utilize lactose as an energy source

Lac Operon: Stepwise Mechanism

Step 1 – Basal Transcription
 Low-level transcription occurs even when the repressor is bound
 Small amounts of permease (lacY) and β-galactosidase (lacZ) are present

Step 2 – Lactose Entry
 Lactose enters the cell through basal levels of permease

Step 3 – Inducer Formation
 Lactose is converted into allolactose by β-galactosidase
 Allolactose acts as the inducer

Step 4 – Repressor Inactivation
 Allolactose binds to the repressor, causing a conformational change
 Repressor can no longer bind to the operator

Step 5 – Transcription Initiation
 RNA polymerase binds to the promoter
 Structural genes lacZ, lacY, and lacA are transcribed

Step 6 – Translation and Function
 mRNA is translated into enzymes
 Permease imports more lactose, β-galactosidase breaks lactose into glucose and galactose
 Allolactose production continues, keeping the operon “on”

Step 7 – Termination
 When lactose is depleted, no allolactose is made
 Repressor binds to the operator again
 Transcription is inhibited

Discovery of Gene Regulation: Jacob and Monod

Key Findings
 Genes can be turned on or off depending on environmental conditions
 A group of genes in the same metabolic pathway can be controlled together by a single regulatory system called an operon

Operon Components
 Structural genes code for enzymes
 Promoter is where RNA polymerase binds
 Operator is a DNA segment that acts as an on/off switch
 Repressor protein binds to the operator to block transcription when the gene is not needed

Mutations
 Studying the lac operon, they discovered mutations in certain genes that can alter how the operon is regulated

Mutations Affecting Lac Operon Regulation

lacI⁻ Mutations
 Occur in the repressor gene
 Prevent production of a functional repressor
 Operon is always on, even without lactose

lacOᶜ Mutations
 Occur in the operator sequence
 Prevent repressor from binding
 Operon is continuously expressed

Significance
 Specific mutations can alter control of gene expression
 Reveals how DNA sequences and regulatory proteins interact
 Explains how mutations in regulatory genes or DNA sites disrupt operon control, a key insight in molecular genetics

Mutations Affecting Lac Operon Proteins

LacZ and LacY Mutations
 Mutations in LacZ or LacY disrupt lactose metabolism
 Affect the function of the proteins
 Do not affect the regulation of their synthesis

Studying Gene Regulation with Mutations

Experimental Approach
 Examined single gene mutations in E. coli
 Crossed mutant cells via conjugation

Purpose
 Applied different combinations of mutations in plasmids or chromosomes
 Determined how the gene was regulated under different genetic conditions

Cis and Trans Regulation of Gene Expression

Cis Regulation
 Controls gene expression only on the same piece of DNA
 Example: promoter sequence of the lac operon acts in cis on lacZ

Trans Regulation
 Controls gene expression on other DNA molecules
 Example: repressor protein acts in trans on lacZ

Partial Diploids
 Bacteria are typically haploid
 A plasmid carrying the lac operon is added, creating a partial diploid
 Not every gene is in the plasmid, allowing study of cis and trans effects

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Cis Regulation

 Cis = DNA part
 It only controls the gene right next to it
 It cannot move

 Example:
  The operator sequence
  If it’s broken, only that operon is affected

Trans Regulation

 Trans = protein
 It can move around the cell
 It can control any copy of the gene

 Example:
  The lac repressor protein (LacI)
  It can bind to operators on ANY DNA

Partial Diploids

 Bacteria get a plasmid with another lac operon
 Now you can see:
  If the mutation only affects its own operon → cis
  If the mutation affects both operons → trans

Notation for Mutations

Mutation Symbols
 Non-functional mutations are designated with a “-” superscript, e.g. lacZ-
 Wild type is designated with a “+” superscript, e.g. lacZ+

Partial Diploid Example
 Bacteria have a mutation in the lacZ gene and wild type lacY in the genome
 Plasmid carries wild type lacZ and mutant lacY
 Genome genotype: lacZ- lacY+
 Plasmid genotype: lacZ+ lacY-
 One good copy of each gene allows the bacterium to metabolize lactose

Trans Dominance of lacI

Partial Diploid Example
 Genotypes: lacI+ lacZ- / lacI- lacZ+
 lacI+ on one DNA molecule, lacI- on the other

Repressor Function
 Repressor gene (lacI) is trans acting and dominant
 Repressor produced by lacI+ can bind to both operators and repress transcription when lactose is absent

Lactose Effect
 When lactose is present, it inactivates the repressor
 Functional β-galactosidase is produced from lacZ
 Bacteria functions normally, like wild type, in the presence or absence of lactose

Super-Repressor Mutation (lacI^s)

Partial Diploid Example
 Genotypes: lacI^s lacZ+ / lacI+ lacZ+
 Super-repressor mutation makes repressor insensitive to allolactose

Repressor Function
 Mutant repressor binds the operator in the presence or absence of allolactose
 Called a super-repressor because it is very dominant
 Blocks transcription regardless of lactose availability

Effect on Lactose Metabolism
 Bacteria cannot metabolize lactose
 Transcription of structural genes is inhibited independent of lactose
 Super-repressor binds both operators and prevents RNA polymerase from initiating transcription

Constitutive Operator Mutation (lacO^C)

Partial Diploid Example
 Genotypes: lacI+ lacO+ lacZ- / lacI+ lacOC lacZ+

Operator Function
 lacOC mutation prevents repressor from binding
 lacO is cis acting, so only affects the genes on the same DNA molecule

Effect on β-Galactosidase Production
 β-galactosidase is produced regardless of lactose presence when paired with lacZ+ on the same DNA
 If lacZ+ were paired with lacO+ on the same sequence, production would occur only in the presence of lactose

Effect of lacOC with Mutant lacZ

Partial Diploid Example
 Genotypes: lacI+ lacO+ lacZ+ / lacI+ lacOC lacZ-

Operator and Protein Function
 lacOC prevents repressor from binding
 Promotes transcription of structural genes on the same DNA

Effect on β-Galactosidase
 With wild-type lacZ, β-galactosidase is produced normally only when lactose is present
 With mutant lacZ-, transcription produces non-functional protein in the presence or absence of lactose

Lac Operon Expression Table

Strain a: lacI lacO lacZ lacY
 Lactose absent: B-gal +, Permease -
 Lactose present: B-gal +, Permease +

Strain b: lacI+ lacO lacZ lacY
 Lactose absent: B-gal +, Permease -
 Lactose present: B-gal +, Permease +

Strain c: lacI lacO lacZ lacY
 Lactose absent: B-gal -, Permease -
 Lactose present: B-gal +, Permease +

Strain d: lacI lacO lacZ lacY / lacI lacO lacZ lacY
 Lactose absent: B-gal +, Permease +
 Lactose present: B-gal +, Permease +

Positive Control and Catabolite Repression

Glucose Preference
 Glucose is the preferred energy source because it requires less energy to metabolize
 Bacteria preferentially metabolize glucose even when other sugars like lactose are present

Catabolite Repression
 When glucose is present, bacteria turn off other metabolic pathways
 This repression of alternative pathways is called catabolite repression
 Example: glucose metabolism represses the lac operon

Positive Control
 When glucose is low, transcription of other metabolic pathways is turned on
 This positive control is independent of the repressor/inhibitor mechanisms

Molecular Mechanism
 Positive control works via catabolite activator protein (CAP) and cAMP
 Levels of cAMP are inversely proportional to glucose levels

CAP and Positive Control of the Lac Operon

CAP Function
 CAP is a helix-turn-helix transcription factor
 Requires cAMP to bind upstream of the lac operon promoter

CAP-cAMP Complex
 CAP-cAMP binds DNA and aids RNA polymerase binding
 Promotes transcription of the lac operon up to 50x

Glucose and cAMP Relationship
 Glucose low → cAMP high
 CAP-cAMP exerts positive control on more than 20 operons in E. coli

Effect on Lac Operon
 When glucose is low and lactose is present, CAP-cAMP binds DNA
 Increases RNA polymerase efficiency
 Results in high rates of transcription and translation of structural genes
 Leads to production of glucose from lactose

Effect of High Glucose on Lac Operon

cAMP Levels
 High glucose → cAMP levels are very low

CAP-cAMP Complex
 Few or no CAP-cAMP complexes form
 Reduced binding of RNA polymerase to lac promoter

Expression Outcome
 Significantly reduced transcription of lac operon
 Lactose metabolism is minimized while glucose is present