Prokaryotic Gene Regulation

• Lecture 7 covers gene regulation in prokaryotes
• The lecture was on February 26th, 2025

Learning Objectives

• Bacteria regulate gene expression to adapt to environmental changes
• Illustrates negative regulation of the lac operon in E. coli
• The discovery and effects of mutants in the lac operon is described
• Compares and contrasts the trp and lac operons

Key Concept: Bacteria as Nutritional Opportunists

• Bacteria are economical and exploit nutritional opportunities

Bacterial Response to Environmental Change

• Bacteria regulate transcription in response to environmental shifts
• Natural selection favors bacteria that produce necessary products
• Bacteria can utilize various nutrients acquired from the environment or synthesized within the cell
• The most efficient bacteria minimize energy expenditure for metabolism

Regulation of Metabolic Pathways

• Two levels of control operate in metabolic pathways
• Enzyme activity undergoes "feedback inhibition" for rapid, short-term adjustments
• Enzyme expression is regulated to control gene expression

Genetic Switches

• Transcription regulation relies on DNA-protein interactions
• Promoters are essential DNA segments for transcription
• Regulatory proteins bind to DNA segments near promoters, acting as genetic switches
• Activators are proteins that enhance transcription, resulting in positive regulation
• Repressors are proteins that inhibit transcription, resulting in negative regulation
• Operators are DNA segments to which repressors bind
• Both activators and repressors are classified as DNA-binding proteins

DNA Binding Proteins (DNABPs)

• DNABPs have two significant sites
• The DNA-binding site interacts directly with specific DNA sequences
• The allosteric site binds an effector molecule, which alters the protein's conformation, causing DNABP to cycle between active/inactive forms

Operon Regulation

• A cluster of functionally related genes under the control of a single operator
• A stretch of DNA consists of the operator, promoter, and the genes they regulate

Operon Control

• Operons can be switched off by a repressor protein thru blocking RNA polymerase
• Repressors are products of regulatory genes

Concept Check: Activators in Prokaryotes vs. Eukaryotes

• Activators in bacteria, which bind to DNA to start transcription, resemble transcription factors in eukaryotes

Key Concept: lac Operon

• The lac operon is inducible

The lac Regulatory Circuit

• The lac operon in E. coli facilitates lactose metabolism
• The lac operon consists of:
  • Promoter (P)
  • Operator (O)
  • Structural genes (Z, Y, A)
  • Regulatory gene (I)

lac Structural Genes and Lactose Metabolism

• Metabolism of lactose needs two enzymes
• Permease transports lactose into the cell
• β-galactosidase modifies lactose into allolactose, and cleaves lactose to yield glucose and galactose

lac System Components

• Three regulatory components:
  • The gene for the lac repressor (lacl or I) is located close to other lac genes
  • The lac promoter site (P)
  • The lac operator site (O)

lac System without Lactose

• The repressor is bound to the operator
• The operon is not transcribed (default state)
• Lactose metabolism doesn't proceed

lac System with Lactose

• Allolactose binds to the repressor
• The repressor is unable to bind to the operator
• The operon is transcribed
• Lactose metabolism proceeds

lac Operon as an Inducible System

• The lac operon is transcribed if lactose is present, making it an inducible operon
• Lactose (actually allolactose) acts as the inducer
• Some other operons are repressible, meaning the end product stops transcription (e.g., trp operon)

Student Activity

• Describe the steps for lactose metabolism when lactose is absent and when lactose is present

Key Concept: lac Operon Mutations

• Mutations in the lac operon can result in varied effects

Discovery of the lac System

• Francois Jacob and Jacques Monod deduced the lac operon's negative regulation in the 1950s via genetic analysis
• They used conjugation in E. coli to study lac operon components
• Bacteria were constructed that were diploid for the lac operon, as they are normally haploid
• Conjugation requires an F plasmid
• Jacob and Monod created F' cells using this process
• F' cells are bacteria which have an F' factor
• Which means they have an F plasmid with some chromosomal genes
• Strains were created with heterozygous genes for selected lac mutations and with a still haploid overall genome
• Made able to analyze if mutations were dominant or recessive

Categories of Regulation

• Trans-acting: Regulatory products that diffuse and interact with DNA molecules.

  • Includes structural genes (Z, Y) and regulatory gene (I).

• Cis-acting: Regulatory sequences only affecting genes on the same molecule where they are found.

  • Includes the promoter (P) and operator (O).

Structural Gene Mutations

• When looking at structural mutations, both Z+ and Y+ (wild type) are dominant to Z- and Y- (inactive), respectively)
• Heterozygotes were lac+ (able to metabolize lactose)

Regulatory Gene Mutations

• Mutations caused a non-functional repressor (I-)
• Unable to bind operator (affecting the DNA binding site)
• Phenotype regarding lactose metabolism: the lac operon genes will be expressed in both the presence and absence of lactose
• Causes constitutive mutations are lac operon genes are continuously expressed with or without an iducer

I+/I- Heterozygotes

• I+/I- heterozygotes retained normal function, with I+ dominant over I-

Regulatory Gene Alterations

• Another mutation caused the repressor to bind to the operator regardless of the presence of an inducer (super repressor, IS)
• The inducer is unable to bind the repressor because the allosteric site of the protein is affected

I+/IS Heterozygotes

• No transcription of structural genes occurred

Operator Mutations

• They prevent the repressor binding and blocking transcription
• Meaning heterozygotes (O+/OC) still transcribe without lactose present
• Leading to constitutive conditions

Promoter Mutations

• Mutations prevent the transcription of operon
• In heterozygotes still function normally

Key Concept: Dual Control of the lac Operon

• The lac operon is under dual positive-negative control

Positive Gene Regulation

• Some operons also subject to positive control thru a transcriptional activator, like cAMP receptor protein (CRP)
• When glucose is scarce, CRP is activated by cyclic AMP (cAMP)
• Activated CRP promoter of lac operon, which increases both affinity of RNA polymerase, and transcription

CRP Regulation with Glucose

• When glucose levels increase, the CRP detaches from the lac operon
• Transcription then returns to a normal rate
• CRP helps regulate other operons encoding catabolic pathway enzymes in E. coli

Key Concept: trp Operon

• The trp operon is repressible

Repressible vs. Inducible Operons

• Inducible operons are usually off, requiring an inducer to inactivate the repressor and start transcription (e.g., lac operon)
• Repressible operons are typically on, but a repressor can bind to the operator to halt transcription (e.g., trp operon)

Operon Function in Metabolism

• Inducible operons operate in catabolic pathways and are induced as needed
• Repressible operons operate in anabolic pathways and are repressed as needed

Repressible Operons

• The repressor can be in either an active or inactive state, based on the presence of certain molecules
• A corepressor is a molecule that partners with a repressor protein to turn off an operon

The trp Operon in E. coli

• E. coli can synthesize tryptophan
• All enzymes needed are encoded by genes in the trp operon
• The trp operon is on by default, allowing tryptophan synthesis
• When tryptophan is present, it binds to the trp repressor protein, which then shuts down the operon
• The repressor is active only when the corepressor tryptophan is present
• The trp operon is repressed when tryptophan levels are high

Concept Check: trp vs. lac Corepressors

• Contrast how binding a trp corepressor affects repressor function and transcription of the trp operon versus how the lac inducer affects the lac repressor

Next Class

• Gene regulation in eukaryotes is covered in Chapter 12