Chapter 11: Microbial Genetics – DNA Replication, Gene Expression, Regulation, Mutations, and Recombination

Chapter 11: Microbial Genetics

Overview

This chapter covers the fundamental processes of microbial genetics, including DNA replication, gene expression (transcription and translation), gene regulation (operons), mutations, and genetic recombination. Understanding these processes is essential for comprehending how genetic information is maintained, expressed, and altered in microorganisms.

DNA Replication

Semiconservative Replication

• Definition: DNA replication is termed semiconservative because each new DNA molecule consists of one parental (original) strand and one newly synthesized strand.

• Process: The parental DNA molecule unwinds, and each strand serves as a template for the synthesis of a new complementary strand.

• Directionality: New DNA strands are synthesized in the 5' to 3' direction.

Origin of Replication

• Definition: The origin of replication is a specific sequence where DNA replication begins.

• AT-rich Regions: The origin is often AT-rich because A-T base pairs are held together by two hydrogen bonds (compared to three in G-C pairs), making them easier to separate.

Replication Fork and Enzymes

• Replication Fork: The Y-shaped region where the DNA is split into two separate strands for copying.

• Key Enzymes and Their Functions:

Enzyme/Factor	Function
Helicase	Unzips the DNA helix by breaking hydrogen bonds between bases.
Topoisomerase (Gyrase)	Relieves supercoiling ahead of the replication fork.
Single-stranded binding proteins	Stabilize unwound DNA strands and prevent re-annealing.
Primase	Synthesizes short RNA primers to provide a starting point for DNA synthesis.
DNA Polymerase III	Main enzyme that adds nucleotides in the 5' to 3' direction.
DNA Polymerase I	Removes RNA primers and replaces them with DNA.
Ligase	Seals gaps between Okazaki fragments on the lagging strand.

Leading and Lagging Strands

• Leading Strand: Synthesized continuously toward the replication fork.

• Lagging Strand: Synthesized discontinuously away from the fork in short segments called Okazaki fragments.

• Reason for Fragments: DNA polymerase can only add nucleotides in the 5' to 3' direction, necessitating discontinuous synthesis on the lagging strand.

Gene Expression: Transcription and Translation

The Central Dogma

• Definition: The flow of genetic information from DNA to RNA to protein.

• Pathway: DNA → RNA → Protein

Transcription

• Definition: The process of synthesizing RNA from a DNA template.

• Stages:

  1. Initiation: RNA polymerase binds to the promoter region (often with the help of a sigma factor in prokaryotes).

  2. Elongation: RNA polymerase adds complementary RNA nucleotides in the 5' to 3' direction.

  3. Termination: RNA polymerase recognizes a termination sequence and releases the RNA transcript.

• Promoter: DNA sequence where RNA polymerase and sigma factor bind to initiate transcription.

• Template Strand: Only one DNA strand is used as a template for RNA synthesis.

Translation

• Definition: The process of synthesizing proteins using the mRNA transcript as a template.

• Key Components: mRNA, ribosomes (rRNA + proteins), tRNA (with anticodons and attached amino acids).

• Stages:

  1. Initiation: Ribosome assembles at the start codon (AUG, which codes for methionine or formyl-methionine in bacteria).

  2. Elongation: tRNAs bring amino acids to the ribosome, matching codons with anticodons, and peptide bonds form between amino acids.

  3. Termination: Occurs when a stop codon (UAA, UAG, UGA) is reached; release factors disassemble the complex.

• Degeneracy of the Code: Most amino acids are encoded by more than one codon, which helps minimize the effects of mutations.

Comparison: Prokaryotic vs. Eukaryotic Gene Expression

Feature	Prokaryotes	Eukaryotes
Transcription & Translation	Simultaneous	Separate (nucleus vs. cytoplasm)
First Amino Acid	Formyl-methionine	Methionine
mRNA Structure	Often polycistronic (multiple proteins)	Monocistronic (one protein)
Introns/Exons	Absent	Present; introns spliced out
mRNA Modifications	None	5' cap and 3' poly-A tail

Gene Regulation: Operons

Operon Structure and Function

• Definition: An operon is a cluster of genes under the control of a single promoter and operator, allowing coordinated regulation.

• Purpose: Allows bacteria to rapidly respond to environmental changes by turning on/off groups of related genes.

Types of Operons

• Inducible Operons: Usually off; turned on by the presence of a substrate (e.g., lac operon for lactose metabolism).

• Repressible Operons: Usually on; turned off by the presence of the end product (e.g., trp operon for tryptophan synthesis).

lac Operon Example

• No Lactose: Repressor binds operator, blocking transcription.

• Lactose Present: Lactose binds repressor, inactivating it; RNA polymerase transcribes genes for lactose metabolism.

Mutations

Definition and Types

• Mutation: A permanent change in the DNA sequence.

• Wild-type: The natural, non-mutated form.

• Mutant: An organism with a mutation, which may affect morphology, metabolism, or resistance.

Type	Description
Silent	Base change does not alter amino acid.
Missense	Base change results in a different amino acid.
Nonsense	Base change creates a stop codon, truncating the protein.
Frameshift	Insertion or deletion shifts the reading frame, often causing widespread changes.

• Spontaneous Mutations: Occur naturally during DNA replication.

• Induced Mutations: Caused by mutagens (chemicals, radiation).

Mutation Repair Mechanisms

• DNA Polymerase Proofreading: Corrects errors during replication.

• Mismatch Repair: Enzymes detect and repair mismatched bases.

• Excision Repair: Damaged DNA is removed and replaced.

Ames Test

• Purpose: Screens chemicals for mutagenic (and potentially carcinogenic) properties using mutant strains of Salmonella typhimurium.

Genetic Recombination

Definition and Importance

• Genetic Recombination: The exchange of genetic material between different DNA molecules, increasing genetic diversity.

• Horizontal Gene Transfer: Transfer of genes between organisms, not by descent.

Mechanisms of Genetic Recombination in Bacteria

Process	Description
Conjugation	Direct transfer of DNA (usually plasmids) via cell-to-cell contact (sex pilus).
Transformation	Uptake of free DNA fragments from the environment by competent cells.
Transduction	Transfer of DNA by bacteriophages (viruses that infect bacteria).

• Generalized Transduction: Any bacterial gene can be transferred.

• Specialized Transduction: Only specific genes near the phage integration site are transferred.

Transposons

• Definition: Segments of DNA that can move from one location to another within the genome ("jumping genes").

• Effects: Can cause mutations or rearrangements, sometimes beneficial, sometimes harmful.

Summary Table: Key Processes and Enzymes

Process	Main Enzyme(s)	Key Features
DNA Replication	DNA polymerases, helicase, ligase, primase	Semiconservative, bidirectional, Okazaki fragments
Transcription	RNA polymerase, sigma factor	Promoter, template strand, 5' to 3' synthesis
Translation	Ribosome (rRNA + proteins), tRNA	Codons, anticodons, start/stop codons
Gene Regulation	Repressors, activators, RNA polymerase	Operons, inducible/repressible systems

Key Equations and Concepts

• Base Pairing Rules: A pairs with T (or U in RNA), G pairs with C.

• Central Dogma Equation:

$\text{DNA} \xrightarrow{\text{transcription}} \text{RNA} \xrightarrow{\text{translation}} \text{Protein}$

• Mutation Rate: Spontaneous mutation rate is approximately $1$in $10^6$ to $10^9$ per gene per generation.

Practice and Application

• Be able to transcribe a DNA sequence to mRNA and translate mRNA to an amino acid sequence using the genetic code.

• Identify types of mutations from DNA or protein sequences.

• Explain the significance of operons in bacterial gene regulation.

• Describe the mechanisms and significance of genetic recombination in bacteria.