12. Regulation of Gene Expression in Bacteria and Bacteriophage

12. Regulation of Gene Expression in Bacteria and Bacteriophage

Overview

Gene expression in bacteria is tightly regulated to ensure that proteins are produced only when needed. This regulation is primarily achieved at the level of transcription, involving interactions between DNA and regulatory proteins. The classic example of such regulation is the lac operon in Escherichia coli, which serves as a model for understanding inducible gene expression systems.

Transcriptional Control of Gene Expression

Constitutive vs. Regulated Transcription

• Constitutive transcription: Certain bacterial genes are transcribed continuously to perform routine cellular functions.

• Regulated transcription: Other genes are transcribed only in response to specific environmental conditions, allowing the cell to adapt to changes.

• Regulation can affect both the initiation and the amount of transcription.

Mechanisms for Regulating Bacterial Gene Expression

Type	Mechanism	Example
Transcriptional Regulation	Inducible transcription	lac operon
Transcriptional Regulation	Repressible transcription	trp operon
Transcriptional Regulation	Attenuation	trp operon, riboswitches
Posttranscriptional Regulation	mRNA destruction	riboswitches
Posttranscriptional Regulation	Translation blockage	antisense RNA, riboswitches

Negative and Positive Control of Transcription

Negative Control

• Involves repressor proteins binding to regulatory DNA sequences (operators) to prevent transcription.

• Example: The lac repressor binding to the lac operator in the absence of lactose.

Positive Control

• Involves activator proteins binding to regulatory DNA sequences (activator binding sites) to initiate transcription.

• Example: The CAP-cAMP complex binding to the lac promoter to enhance transcription when glucose is absent.

Repressor Proteins and Allostery

Structure and Function

• Repressor proteins have two active sites:

  • DNA-binding domain: Binds to operator or regulatory DNA sequences.

  • Allosteric domain: Binds small molecules (inducers or corepressors) that alter the conformation and function of the DNA-binding domain (allostery).

• Allostery can either inactivate or activate the DNA-binding domain, depending on the molecule bound.

Modes of Allosteric Regulation

• Inducer binding: Inactivates the DNA-binding domain, preventing the repressor from binding DNA (e.g., allolactose in the lac operon).

• Corepressor binding: Activates the DNA-binding domain, enabling the repressor to bind DNA (e.g., tryptophan in the trp operon).

Activator Proteins and Positive Control

Mechanism

• Activator proteins bind to activator binding sites on DNA, facilitating RNA polymerase binding and transcription initiation.

• They also have a DNA-binding domain and an allosteric domain.

Modes of Action

• Activator is inactive until an allosteric effector compound binds, activating the DNA-binding domain.

• Alternatively, an inhibitor can bind the allosteric domain, inactivating the DNA-binding domain.

Regulatory DNA-Binding Proteins: Structure and Interaction

Protein-DNA Interactions

• DNA-binding proteins recognize specific DNA sequences via amino acid side chains that interact with nucleotide bases.

• Specificity is determined by hydrogen, nitrogen, and oxygen atom patterns in the DNA.

• Common structural motif: Helix-turn-helix (HTH), where two α-helices interact with inverted repeats in DNA.

Protein Complexes

• Regulatory proteins may function as monomers, dimers (homodimers or heterodimers), trimers, or tetramers.

• Each polypeptide may interact with a repeat sequence in the DNA.

The lac Operon: An Inducible System

Operon Structure and Function

• An operon is a cluster of genes under coordinated transcriptional regulation by a shared regulatory region.

• The lac operon of E. coli contains:

  • Regulatory region: Promoter (binds RNA polymerase), Operator (lacO) (binds lac repressor), CAP binding site (binds CAP-cAMP complex).

  • Structural genes: lacZ (β-galactosidase), lacY (permease), lacA (transacetylase).

• Transcribed as a single polycistronic mRNA.

Lactose Metabolism and Induction

• Glucose is the preferred energy source; lactose is used only when glucose is absent.

• Lactose is a disaccharide (glucose + galactose) joined by a β-galactoside linkage.

• β-galactosidase breaks lactose into glucose and galactose; permease imports lactose into the cell.

• Allolactose, a lactose isomer, acts as the inducer by binding the lac repressor.

lacI Gene and Repressor Protein

• lacI is adjacent to, but not part of, the lac operon; it encodes the lac repressor protein (a homotetramer).

• The repressor binds to lacO and blocks transcription in the absence of lactose.

• Binding of allolactose to the repressor's allosteric domain inactivates its DNA-binding ability, allowing transcription.

Regulation of the lac Operon

• Negative control: In the absence of lactose, the repressor binds to the operator, preventing transcription.

• Induction: When lactose is present (and glucose is absent), allolactose binds the repressor, which releases from the operator, allowing transcription.

• Basal transcription: Even with the repressor inactivated, only low levels of transcription occur unless positive control is also present.

• Positive control: The CAP-cAMP complex binds to the CAP site, enhancing RNA polymerase binding and transcription when glucose is absent.

• Catabolite repression: When glucose is present, cAMP levels are low, CAP-cAMP does not form, and lac operon transcription is inefficient.

Summary Table: lac Operon Regulation

Condition	Repressor	CAP-cAMP	Transcription Level
No lactose, glucose present	Bound to operator	Absent	None
Lactose present, glucose absent	Released (inducer bound)	Present (high cAMP)	High
Lactose and glucose present	Released (inducer bound)	Absent (low cAMP)	Low (basal)

Key Terms and Concepts

• Operator: DNA sequence where repressor binds.

• Promoter: DNA sequence where RNA polymerase binds to initiate transcription.

• Inducer: Small molecule (e.g., allolactose) that inactivates the repressor.

• CAP (Catabolite Activator Protein): Protein that, when bound to cAMP, activates transcription of the lac operon.

• cAMP (cyclic AMP): Small molecule whose concentration is inversely related to glucose levels; required for CAP binding.

• Polycistronic mRNA: Single mRNA molecule encoding multiple proteins.

Example: The lac Operon in Action

• When lactose is absent: The lac repressor binds to the operator, blocking transcription (negative control).

• When lactose is present and glucose is absent: Allolactose binds the repressor, which releases from the operator; CAP-cAMP binds the promoter, and transcription is highly activated (positive control).

• When both lactose and glucose are present: The repressor is inactive, but CAP-cAMP does not form, so only basal transcription occurs (catabolite repression).

Additional info: The lac operon is a foundational model for understanding gene regulation in prokaryotes and has been instrumental in the development of molecular genetics. The principles learned from the lac operon apply broadly to other inducible and repressible systems in bacteria.