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 via operons, mutations, and genetic recombination. These topics are essential for understanding how microorganisms inherit, express, and alter their genetic information.

DNA Replication

Overall Replication Process

DNA replication is the process by which a cell duplicates its DNA, ensuring genetic continuity between generations. In prokaryotes, replication occurs on both strands simultaneously and follows a semiconservative model.

• Semiconservative replication: Each new DNA molecule consists of one parental (old) strand and one newly synthesized strand.

• Uncoiling: The parent DNA molecule is uncoiled, and the two strands are separated, exposing the nucleotide sequence to serve as templates.

• Template-directed synthesis: New complementary strands are synthesized by using each single-stranded template as a pattern.

Example: The diagram shows parental DNA strands separating and serving as templates for new strands.

Key Events in Prokaryotic DNA Replication

• Origin of replication: Prokaryotic chromosomes have a specific site (AT-rich) where replication begins. Less energy is required to separate AT pairs due to fewer hydrogen bonds.

• Replication forks: Two replication forks form, each with its own set of replication enzymes.

Molecular Machinery Involved in Bacterial DNA Replication

Factor/Enzyme	Function
Topoisomerase	Relaxes supercoiled DNA to make it accessible; relieves stress during unwinding.
Helicase	Unzips the DNA helix by breaking hydrogen bonds between bases.
Primase	Synthesizes RNA primers needed to start replication.
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.
Single-stranded binding proteins	Prevent reformation of double-stranded DNA.
Topoisomerase IV	Separates concatenated chromosomes after replication.

Leading vs. Lagging Strand Synthesis

• Leading strand: Synthesized continuously in the 5' to 3' direction toward the replication fork.

• Lagging strand: Synthesized discontinuously in short segments (Okazaki fragments), each initiated by an RNA primer.

Key Point: DNA polymerase can only add nucleotides in the 5' to 3' direction, necessitating discontinuous synthesis on the lagging strand.

Gene Expression: Transcription and Translation

Central Dogma of Molecular Biology

The central dogma describes the flow of genetic information: DNA → RNA → Protein. This pathway is conserved in all cellular life forms.

• Replication: DNA is duplicated for inheritance.

• Transcription: DNA is used as a template to make RNA.

• Translation: RNA is used to synthesize proteins.

Transcription

Transcription is the process of synthesizing an RNA molecule using DNA as a template.

• Initiation: RNA polymerase binds to the promoter region (with help from sigma factor) and unwinds DNA.

• Elongation: RNA polymerase adds nucleotides complementary to the DNA template strand (5' to 3' direction). Uracil (U) pairs with adenine (A).

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

Key Components: Promoter, leader sequence, coding region, terminator.

Translation

Translation is the process by which ribosomes synthesize proteins using mRNA as a template.

• Start codon: AUG (methionine); in bacteria, the first amino acid is formyl-methionine.

• Codons: Triplets of nucleotides in mRNA, each specifying an amino acid.

• tRNA: Acts as a translator, matching its anticodon to mRNA codons and carrying the corresponding amino acid.

• rRNA: Structural and catalytic component of ribosomes.

Stages: Initiation, elongation (peptide bond formation and translocation), termination (stop codons: UAA, UAG, UGA).

Polyribosomal Complexes

In prokaryotes, transcription and translation can occur simultaneously, allowing rapid response to environmental stimuli.

Comparison: Prokaryotic vs. Eukaryotic Gene Expression

Feature	Prokaryotes	Eukaryotes
Transcription/Translation	Simultaneous	Separate (nucleus vs. cytoplasm)
First amino acid	Formyl-methionine	Methionine
mRNA structure	Polycistronic (multiple proteins)	Monocistronic (single protein)
Introns	Absent	Present (spliced out)
mRNA modifications	None	5' cap, 3' poly-A tail

Gene Regulation: Operons

Operon Structure and Function

Operons are clusters of genes regulated as a single unit, allowing coordinated expression in response to environmental changes.

• Inducible operons: Turned ON by substrate (e.g., lac operon for lactose metabolism).

• Repressible operons: Turned OFF by product (e.g., arginine operon for amino acid synthesis).

Lac Operon (Inducible)

• Regulator: Codes for repressor protein.

• Control locus: Promoter and operator regions.

• Structural locus: Genes for enzymes (β-galactosidase, permease, transacetylase).

• Mechanism: In absence of lactose, repressor binds operator and blocks transcription. Lactose (inducer) binds repressor, inactivating it and allowing transcription.

Arginine Operon (Repressible)

• Default state: ON (repressor inactive).

• Excess arginine: Arginine binds repressor (corepressor), activating it to block transcription.

Mutations

Types and Causes

• Mutation: Permanent change in DNA sequence.

• Wild-type strain: Natural, non-mutated organism.

• Mutant strain: Organism with a mutation, possibly affecting morphology, nutrition, resistance, etc.

• Spontaneous mutations: Random errors during replication.

• Induced mutations: Caused by mutagens (chemical or radiation).

Types of Mutations

Type	Description
Point mutation	Substitution of a single base.
Silent mutation	No effect on protein sequence.
Missense mutation	Changes one amino acid.
Nonsense mutation	Converts codon to stop codon, truncating protein.
Frameshift mutation	Insertion/deletion shifts reading frame, often causing multiple errors.
Back-mutation	Reversion to original sequence.

Mutation Detection and Repair

• Ames test: Detects mutagenic potential of chemicals using Salmonella typhimurium auxotrophs.

• DNA repair mechanisms: DNA polymerase proofreading, mismatch repair, photoreactivation, excision repair.

Effects of Mutations

• Most mutations are harmful or neutral; some confer advantages and drive evolution.

Genetic Recombination

Definition and Mechanisms

Genetic recombination is the process by which DNA from different sources is combined, increasing genetic diversity.

• Horizontal gene transfer: Movement of genetic material between organisms, not by descent.

• Fates of donor DNA: Stable integration, partial integration, degradation, or maintenance as plasmid.

Mechanisms of Genetic Recombination in Bacteria

Process	Description
Conjugation	Direct transfer of plasmid or chromosomal DNA via cell-to-cell contact (sex pilus).
Transformation	Uptake of free DNA fragments from the environment by competent cells.
Transduction	Transfer of DNA via bacteriophage (virus).

• Generalized transduction: Random host DNA fragments are packaged into phage particles.

• Specialized transduction: Specific host genes are transferred by phage.

Transposons

Transposons are DNA segments capable of moving within the genome, causing genetic rearrangements. They can be beneficial or harmful.

Additional info: The notes include humor and memes to aid memory, but all scientific content is relevant for college-level microbiology.