Chapter 9: An Introduction to Microbial Genetics

Introduction to Genetics and the Genome

• Genetics Definitions: Genetics is the science of heredity. It investigates four primary areas:

  • Transmission of biological traits from parents to their offspring.

  • The expression and variation of specific traits.

  • The structure and function of genetic material.

  • The mechanisms by which genetic material changes.

• The Genome: The sum total of all genetic material within a cell, primarily composed of DNA.

  • The majority of genetic material is organized into chromosomes.

  • Non-chromosomal genetic locations include mitochondria, chloroplasts, and plasmids.

  • Genomes of cells always consist of DNA, whereas viral genomes may consist of either DNA or RNA.

• Chromosomes: Discrete cellular structures consisting of neatly packaged DNA molecules.

  • Eukaryotic Chromosomes: Located in the nucleus; characteristically multiple and linear in structure.

  • Bacterial Chromosomes: Characteristically a single circular loop.

• Genotypes vs. Phenotypes:

  • Genotype: The sum of all gene types that constitute an organism's genetic makeup.

  • Phenotype: The observable traits created by the expression of the genotype.

• Genes: The fundamental unit of heredity responsible for specific traits. Formally defined as a site on a chromosome providing information for a cell function, or a DNA segment containing the code for a protein or RNA molecule.

  • Structural Genes: Code for proteins.

  • RNA Genes: Code for various RNA molecules.

  • Regulatory Genes: Control the expression of other genes.

• Genome Size Comparisons:

  • Smallest virus: $4$ to $5$ genes.

  • $E. coli$: A single chromosome containing $4,288$ genes.

  • Human cell: $46$ chromosomes containing approximately $31,000$ genes.

DNA Structure and Packaging

• Packaging and Coiling:

  • Prokaryotes: DNA is compacted via supercoils or superhelices by the enzyme DNA gyrase, which introduces reversible twists to create a tight bundle.

  • Eukaryotes: Packaging involves complex levels of coiling. It starts with nucleosomes, which are DNA strands wrapped around histone proteins. These further condense into chromatin fibers and metaphase chromosomes.

• Chemical Structure of DNA: The basic unit is the nucleotide, comprising:

  • A deoxyribose sugar.

  • A phosphate group.

  • A nitrogenous base: Adenine ($A$), Guanine ($G$), Thymine ($T$), or Cytosine ($C$).

• Backbone and Pairing:

  • Nucleotides bond covalently to form a sugar-phosphate backbone.

  • Bases span the center of the molecule to pair with complementary strands through hydrogen bonding:

    • Adenine ($A$) pairs with Thymine ($T$) via $2$ hydrogen bonds.

    • Guanine ($G$) pairs with Cytosine ($C$) via $3$ hydrogen bonds.

• Antiparallel Arrangement: The two strands run in opposite directions; one is oriented $5'$ to $3'$ and the other is $3'$ to $5'$.

• Significance of Structure:

  • Code Maintenance: Base pairing constancy ensures the code is retained during reproduction.

  • Variety: The specific order of bases determines the sequence of RNA and proteins, leading to diverse phenotypes.

The DNA Replication Process

• Semiconservative Replication: Each daughter DNA molecule consists of one original parent strand and one newly synthesized strand.

• Replication Origins: Replication starts at a specific site called the origin of replication, which is typically AT-rich, requiring less energy to separate the strands.

• Bidirectional Growth: Two replication forks move in opposite directions from the origin and meet at a termination site.

• Enzymes of Replication:

  • Helicase: Separates the two DNA strands.

  • Primase: Synthesizes an RNA primer to initiate synthesis.

  • DNA Polymerase III: Adds bases to the new DNA chain and performs proofreading.

  • DNA Polymerase I: Removes RNA primers, fills gaps between fragments with DNA nucleotides, and repairs mismatches.

  • Ligase: Performs final binding of nicks in the DNA during synthesis and repair.

  • Gyrase: Re-initiates supercoiling after replication.

• Leading vs. Lagging Strands:

  • Leading Strand: Synthesized continuously in the $5'$ to $3'$ direction.

  • Lagging Strand: Synthesized discontinuously in short segments called Okazaki fragments, each requiring multiple RNA primers.

Transcription: From DNA to RNA

• Central Dogma: Genetic information flows from DNA (Replication) to RNA (Transcription) to Protein (Translation).

• RNA Features:

  • Single-stranded molecule capable of complex secondary and tertiary folding.

  • Contains Uracil ($U$) instead of Thymine ($T$).

  • Contains ribose sugar instead of deoxyribose.

• Major Types of RNA:

  • Messenger RNA (mRNA): Carries the master DNA code to the ribosome; contains triplet codes called codons.

  • Transfer RNA (tRNA): Acts as a translator; cloverleaf structure with an anticodon loop at one end and an amino acid binding site at the other.

  • Ribosomal RNA (rRNA): Forms the physical structure of ribosomes and facilitates protein synthesis.

  • Primer: An RNA segment that initiates DNA replication.

• Stages of Transcription:

  1. Initiation: RNA polymerase, guided by a sigma factor, binds to the promoter region of the DNA and unwinds the helix. The template strand runs in the $3'$ to $5'$ direction.

  2. Elongation: RNA polymerase adds complementary nucleotides (using $U$ for $A$) in the $5'$ to $3'$ direction. Transcripts are generally $100$ to $1,200$ bases long.

  3. Termination: The enzyme recognizes a termination sequence and releases the mRNA transcript.

Translation: From RNA to Protein

• The Master Genetic Code: Represented by $64$ mRNA codons. The code is universal and redundant (multiple codons may specify the same amino acid).

  • Start Codon: Typically AUG (codes for Methionine).

  • Stop Codons: UAA, UAG, and UGA (no corresponding tRNAs).

• Ribosome Anatomy: Prokaryotic ribosomes are $70S$. The large subunit has three binding sites:

  • P (Peptidyl) site: Holds the tRNA with the growing polypeptide chain.

  • A (Aminoacyl) site: Binds the next incoming tRNA carrying an amino acid.

  • E (Exit) site: Where empty tRNAs are discharged.

• Translation Steps:

  1. Ribosome scans mRNA for the start codon ($AUG$).

  2. A tRNA with the complementary anticodon ($UAC$) enters the P site.

  3. A second tRNA enters the A site.

  4. A peptide bond forms between the two amino acids.

  5. Translocation: The ribosome shifts, discharging the first tRNA and moving the second into the P site.

  6. The process repeats until a stop codon is reached.

• Polyribosomal Complex: An assembly line of multiple ribosomes reading a single mRNA simultaneously for mass protein production.

Genetic Variations and Eukaryotic Differences

• Eukaryotic Gene Expression vs. Prokaryotic:

  • In eukaryotes, transcription occurs in the nucleus and translation in the cytoplasm (not simultaneous).

  • Eukaryotic mRNA is monocistronic (codes for one protein), whereas bacterial mRNA often codes for multiple.

  • Eukaryotic DNA contains introns (non-coding sequences) and exons (coding sequences).

  • Splicing: Spliceosomes remove introns and join exons before the mRNA leaves the nucleus.

• Operons (Prokaryotic Regulation):

  1. Inducible Operons (lac operon): Normally OFF. Turned ON by a substrate (inducer). Lactose binds to the repressor, causing it to release the operator so RNA polymerase can transcribe genes for lactose metabolism ($b$-galactosidase, permease).

  2. Repressible Operons (arg operon): Normally ON. Turned OFF when the product (arginine) accumulates. Arginine acts as a corepressor, activating the repressor to bind the operator and block further synthesis.

• Regulatory RNAs:

  • Riboswitch: A segment of mRNA that regulates its own translation.

  • RNA Interference: Includes miRNA, siRNA, and antisense RNA that regulate eukaryotic expression.

Mutations: Changes in the Code

• Definitions: A mutation is a change in the nitrogen base sequence of DNA (genotype change leading to phenotype change). Wild type is the natural state; mutant strain shows variance.

• Categories of Mutations:

  • Point Mutation: Addition, deletion, or substitution of a few bases.

  • Missense: Changes one amino acid; effects vary.

  • Nonsense: Changes a normal codon to a stop codon; usually severe.

  • Silent: Base change that does not alter the amino acid.

  • Back-mutation: A mutated gene reverses to its original state.

  • Frameshift: Insertion or deletion of bases that shifts the reading frame; almost always results in a nonfunctional protein.

• Causes and Repair:

  • Spontaneous: Errors in replication.

  • Induced: Exposure to mutagens (e.g., Nitrous acid, Ethidium bromide, UV radiation which causes pyrimidine cross-links, X-rays which break DNA).

  • Repair Mechanisms: DNA polymerase proofreading, mismatch repair, light repair (UV), and excision repair.

• Ames Test: Uses Salmonella enterica (histidine mutant) to screen chemicals for mutagenic potential. High rates of back-mutation indicate a strong mutagen.

DNA Recombination and Viruses

• Bacterial Recombination:

  • Conjugation (Direct): DNA transfer via a pilus. Requires an F factor (fertility plasmid). Hfr cells transfer chromosomal DNA.

  • Transformation (Indirect): Uptake of free DNA fragments from a lysed donor cell by a competent recipient (e.g., Griffith’s mouse experiment with capsule genes).

  • Transduction (Indirect): DNA transfer via a bacteriophage.

    • Generalized: Random fragments of host DNA are packaged.

    • Specialized: Specific parts of the host genome are incorporated.

  • Transposons: "Jumping genes" that move from one genomic site to another.

• Viral Genetics:

  • Viral genomes are minimalist, containing only genes for replication and host infection.

  • DNA Viruses: Replication usually occurs in the host nucleus. Viral DNA is transcribed into mRNA, then translated on host ribosomes.

  • RNA Viruses: Replication usually occurs in the host cytoplasm. Positive-strand RNA viruses can be translated directly into viral proteins.

  • All viruses rely on host ribosomes and tRNAs for protein synthesis.