Week 3 S - Gene Regulation in Prokaryotes

Molecular Genetics: Gene Regulation in Prokaryotes

Overview

• Gene Regulation Importance:
  • Allows rapid responses to environmental changes.
  • Enables bacteria to adapt their physiology to meet current requirements.
  • Ensures economical deployment of cellular resources.
  • Optimizes gene expression due to strong evolutionary pressure.

Control Points in Gene Expression

• Three major control points:
  • Transcription: The main control point in bacteria, primarily at initiation.
  • Transcript processing.
  • Translation.

The Operon Model

• Discovery: Jacques Monod and Francoise Jacob (1961).
• Components:
  • Regulatory gene: Encodes a regulatory protein.
  • DNA: Contains cis-acting elements.
  • mRNA: Messenger RNA.
  • Regulatory protein: A trans-acting product.
  • Structural gene: Encodes a protein product.
• Function:
  • Controls gene expression (on/off).
  • Promoters and terminators are examples of cis-acting elements.

Negative Control

• Mechanism:
  • A repressor protein binds to the operator, preventing gene transcription.
  • In the absence of the repressor, RNA polymerase can transcribe the structural gene.
  • Repressor blocks RNA polymerase.

Positive Control

• Mechanism:
  • A transcription factor binds to a cis-element (binding site), which allows recruitment of RNA polymerase onto the promoter, initiating transcription.
  • Transcriptional activator.

Operons in Bacteria

• Organization:
  • Multiple genes under one promoter, resulting in one transcript (polycistronic transcript).
  • Genes often have a common function (e.g., same metabolic pathway).
  • Includes enzymes, transporters, and regulators.
• Advantage:
  • Allows coordinated gene expression where genes are switched on and off together as a group.
  • Example: The lac operon.

The lac Operon

• Components:
  • lacI: Encodes the repressor.
  • lacZ: Encodes β-galactosidase.
  • lacY: Encodes permease.
  • lacA: Encodes transacetylase.
  • Operator/promoter region.
  • Transcription start and termination sites.
• Regulation:
  • lacI is transcriptionally independent of lacZYA.
  • lacZYA are under negative control, with the default state being 'on'.
  • The operon is switched off by a repressor.
  • Not an absolute on/off switch; normal changes are 5-100 fold.
• LacI Characteristics:
  • Homotetramer (38 kDa subunits).
  • Approximately 10 tetrameric LacI molecules per cell.

lacZYA Promoter Region

• Key Features:
  • Transcription start site for lacZYA.
  • Promoter region (RNA polymerase binding site).
  • Operator region (LacI binding region).
  • DNA 'melting' region.

On and Off States of lacZYA

• Induction:
  • Induction of lacZYA leads to an increase in β-Galactosidase from 5 to ~5000 copies/cell.
  • Expression commences in 2-3 minutes.
  • β-Gal can make up to 10% of soluble cellular protein.
• Reversibility:
  • Synthesis is reversible depending on the presence or absence of an inducer.
  • Stable mRNA with a half-life ( t_{1/2} ) of ~3 minutes.
• Basal Expression:
  • β-Gal 'basal' expression occurs even without an inducer.

LacI and Allolactose

• Inducer Specificity:
  • LacI recognizes allolactose, which is an isomer of lactose, rather than lactose itself.
  • Lactose = galactose-(\beta1->4)-glucose.
  • Allolactose = galactose-(\beta1->6)-glucose.
  • β-Gal converts lactose to allolactose during a low-level side reaction.

IPTG - Gratuitous Inducer

• Function:
  • Isopropylthiogalactoside (IPTG) acts like allolactose on LacI but is not degraded by β-gal enzyme.
• Key Point:
  • Both the regulator and the enzyme recognize the same substrate.

LacI Activity

• Regulation:
  • LacI is active in the absence of an inducer, preventing lacZYA expression.
  • In the presence of an inducer, LacI becomes inactive, allowing lacZYA expression.
• Mechanism:
  • Inducer binds to LacI, causing a conformational change and loss of affinity for the operator.

Two Roles for LacI

• Functions:
  • Recognition of the inducer (allolactose).
  • Repression of transcription.
• Binding Sites:
  • Lac operon operator.
  • Inducer.
• Allosteric Control:
  • Change in 'shape' upon inducer binding.
  • Results in a loss of activity for the operator-DNA binding site.
  • DNA binding capacity is switched on/off by occupation of the inducer-binding site.

LacI - Subunit Organization

• Domains:
  • DNA-binding domain.
  • Tetramerization domain.
  • Inducer-binding region (including core subdomains 1 and 2).

LacI - Domain Function

• DNA-binding Domain:
  • Contains a helix-turn-helix motif.
  • Fits into the major groove of DNA.
  • Makes specific contacts.
• Core Subdomains:
  • Similar structures.
  • Composed of a 6 \beta-stranded sheet with 2 helices on either side.
  • Enable dimerization.

lac Operator

• Structure:
  • Palindrome (inverted repeat).
  • Each repeat represents an operator half-site.
  • Symmetry of the operator is reflected in the symmetry of the LacI dimer (two DNA binding domains/dimer).
• Binding Affinity:
  • Mutations that improve symmetry increase LacI binding (x10).

Allosteric Change in Conformation

• Inducer Effect:
  • Inducer binding causes a massive impact on affinity.
  • Two HTHs insert into consecutive major grooves.
  • DNA is bent upon binding by ~45 degrees.
  • This is a common feature of DNA binding.
• Mechanism:
  • Inducer stimulates a change in LacI structure (headpiece re-orientation).
  • Results in an inability to bind the operator and hinge disruption.

LacI - Why Tetrameric?

• Function:
  • Tetramer can bind two operators simultaneously.
• Additional Operators:
  • Two other, weaker operators: O2 & O3.
  • Strong operator: O1.
• Significance:
  • Loss of both O2/O3 reduces repression by x50.
  • LacI must bind O1 and O2 or O3 for strong repression.

LacI and Repression Mechanism

• Binding Effects:
  • LacI and RNA polymerase can bind together at the lac P/O region.
  • LacI binding improves RNA polymerase binding.
• Repression:
  • RNA polymerase cannot initiate transcription when in complex on the promoter with LacI.
  • LacI causes RNA polymerase to be 'locked' within a lac promoter complex.
• Induction:
  • Inducer releases LacI, allowing RNA polymerase to initiate transcription.
  • In most cases, repressors work by promoter occlusion.

Catabolite Repression

• Second Layer of Control:
  • Lactose cannot induce lac if glucose is present.
  • Glucose is preferred because it is a better energy source than lactose.
• Mechanism:
  • Caused by a global regulatory control system.
  • Cyclic AMP (cAMP) and CRP (cAMP receptor protein) involved.
  • Approximately 20 operons in E. coli are controlled this way.

Cyclic AMP - '2nd Messenger'

• Function:
  • Catabolite repression is imposed via cAMP.
  • CRP (also known as CAP - Catabolite Activator Protein) is a positive regulatory protein.
• Dual Control:
  • lac operon is under dual control by LacI and CRP.
• Promoter Activity:
  • lac promoter is a poor promoter with very weak activity.
  • Requires 'assistance' to exhibit strong activity.

CRP Regulation

• Activation:
  • Switches genes on and assists transcription initiation.
  • Only active when bound to cAMP; cAMP acts as a typical co-activator molecule.
• Glucose Effect:
  • Adenylate cyclase (ATP → cAMP) is inhibited by high glucose levels.
  • High glucose leads to low cAMP, and low glucose leads to high cAMP.

cAMP and Glucose Effects on lacZ Expression

• Low Glucose:
  • High cAMP levels.
  • CRP/cAMP complex forms and binds to the CRP-binding site, recruiting RNA polymerase to the promoter.
  • Results in expression of lacZ.
• High Glucose:
  • Low cAMP levels.
  • CRP remains inactive with weak DNA binding affinity.
  • RNA polymerase cannot bind the lac promoter unless CRP assists, resulting in little to no expression.

CRP Structure and Function

• Structure:
  • Homodimer.
  • Allosteric activation by one cAMP molecule.
• Subunits:
  • Each subunit contains a cAMP binding site, a DNA-binding domain, and a transcription-activating region.
• DNA Binding:
  • Dimer binds ~22 bp DNA, recognizing a 10 bp palindromic sequence within the 22 bp site.

CRP Binding Site

• Features:
  • CRP prefers to bind to a site with two inverted pentameric motifs (TGTGA).
  • Specific spacing (bp gap) is important, forming a 'hyphenated' palindrome.
• Binding Mode:
  • Allows both subunits to bind, one to each half site.

CRP-Induced DNA Bending

• Effect:
  • Causes a severe bend of ~90 degrees in the DNA.
• Purpose:
  • The exact purpose of the bend is unclear.

CRP Binding Site Locations

• Variations:
  • CRP binds at different locations relative to the promoter, depending on the operon.
• Examples:
  • lac: Adjacent and just upstream of the promoter.
  • gal : Within the promoter.
  • Class I: CRP binds long way upstream of promoter.

CRP Activation - Class I and II

• Class I:
  • C-terminus of alpha subunit of RNA polymerase is bound.
  • Rate of promoter binding is increased (closed complex formation).
• Class II:
  • C and N-termini of alpha subunit, plus sigma factor, may be bound.
  • Rate of isomerization of promoter from closed to open complex is increased.

Key Takeaways

• Regulation:
  • Interaction between cis and trans-acting elements.
  • Transcription regulation involves interactions at/around the promoter.
  • Inactive genes can be induced; active genes can be repressed.
• Regulators:
  • One regulator may have many targets.
  • Regulators make sequence-specific interactions with DNA.
• Small Molecules:
  • Small molecules (co-effectors) often control the activity of regulators.
• Allosteric Changes:
  • Allosteric changes induce conformational effects that adjust regulator DNA binding.
• Cooperation:
  • Regulators can cooperate to exert control in response to multiple effects.

Lac Regulation - Additional Information

• Extra details available for interested learners.

LacI Tetramer Bound to DNA

• Visual representation of LacI tetramer binding to DNA.

lac Operon Regulation Diagrams

• Diagrams illustrating different scenarios of lac operon regulation based on the presence or absence of lactose and glucose.

Common Features of Transcription Control

• Components:
  • Trans-acting regulatory proteins and effector molecules.
  • Binding to cis-elements.
• Cis-Elements:
  • Near the promoter, upstream of the gene.
• Regulatory Proteins:
  • Bind to specific sequence motifs in DNA (cis-elements).
  • Contact region can be larger than 10 bp, involving non-specific interactions.

Operon Consequences

• Coordinated Expression:
  • All genes in the operon are switched on/off together.
• Induction Order:
  • Order of protein appearance depends on the gene order in the operon.
  • β-gal appears first, followed by permease, then transacetylase.
• Protein Proportions:
  • Relative proportions of the three proteins are similar due to the common transcript.
• Regulator Placement:
  • Regulators are often last in the operon.
  • Most important genes are often first.

Lac Operon Paradox

• Problem:
  • LacY is required for lactose entry, and LacZ is required for allolactose generation.
  • How can the lac operon be induced if LacZ and LacY are not already present?
• Solution:
  • Low-level basal expression (0.1% of fully induced level) enables initial induction.

Mutations of the lac Operator

• Critical Sites:
  • Although LacI contacts 26 bp of DNA, only 8 bp are critical for interaction.
• Effect of Mutations:
  • Mutations in these 8 sites can lead to constitutive expression.

LacI Mutations

• Location Correlation:
  • Mutations affecting specific aspects of LacI function map to the same regions.
• Functional Impacts:
  • Mutations in the DNA-binding domain prevent operator binding.
  • Mutations in the inducer-binding region prevent inducer binding or response.

LacI Affinity

• Operator Affinity:
  • LacI has 10^7 greater affinity for O1 than for genomic DNA.
  • 96% occupancy for O1 at 10 tetramers per cell.
• Inducer Effect:
  • Inducer causes LacI affinity to drop to 10^4 times that of genomic DNA.
  • Occupancy drops to 3%.
• Cellular Distribution:
  • 99.9% of LacI is bound to DNA, either at the specific operator site or at low-affinity non-specific sites.
  • Thus, there is virtually no 'free' LacI in the cell.

Regulatory States

• Repression → Induction:
  • Repressor is deactivated by an inducer.

Constitutive Mutants

• Operator Mutations:
  • Mutations in the operator DNA can result in loss of LacI binding (cis-acting).
  • Cannot be complemented by providing a wild-type copy of the mutated lac operon (cis-dominant).
• lacI Gene Mutations:
  • Mutations in the lacI gene prevent LacI from binding to the operator (trans-acting).
  • Can be complemented by a wild-type lacI gene.

LacI - A Dimer of Dimers

• Structure:
  • Subunits form a dimer through core region interactions.
  • C-terminal helix tetramerizes through 2 Leu heptad repeats.
  • There are 2 DNA-binding sites and 4 inducer-binding sites per tetramer.

Induction & Repression**

• Induction – gene switched on by a signal (often a cognate substrate)
• Repression – gene switched off by signal (often corresponding product)
• Co-effectors: inducers and co-repressors – small molecules/ chemicals that cause expression or repression of specific genes

Class I and II binding sites

• All CRP-dependent promoters have poor -35 sites, and may have poor -10s also – CRP converts poor promoters to stronger promoters
  • Class I – at ~ -61 (separate from promoter)
  • Class II – at ~ -41 (promoter overlap)
  • Both cases – CRP is thought to bind to same face of duplex as RNA pol