Microbial Genetics

Griffith's Experiment & Transformation

Griffith's experiment in 1928 demonstrated that bacteria can acquire new traits by absorbing external genetic material, a process called transformation. This experiment laid the foundation for understanding genetic inheritance in microbes.

• Transformation: Uptake of external DNA by a cell, resulting in a genetic and phenotypic change.

• Example: Griffith showed that non-virulent bacteria could become virulent when mixed with heat-killed virulent bacteria.

Additional info: Later work by Avery, MacLeod, and McCarty identified DNA as the transforming substance.

The Hershey-Chase Experiment

In 1952, Hershey and Chase used bacteriophages to confirm that DNA is the genetic material, not protein.

• Bacteriophage: A virus that infects bacteria, consisting of a protein coat and DNA core.

• Experiment: Radioactive labeling showed that only DNA entered the bacterial cell during infection.

Chargaff's Rules

Erwin Chargaff discovered that DNA composition varies between species, but the amount of adenine (A) equals thymine (T), and cytosine (C) equals guanine (G).

Discovery of DNA Structure

Rosalind Franklin's X-ray diffraction images and Watson & Crick's model revealed the double helix structure of DNA, with base pairing between A-T and G-C.

• Base Pairing: A pairs with T, G pairs with C.

• Antiparallel Strands: DNA strands run in opposite directions.

Detailed DNA Structure

DNA consists of two antiparallel strands of nucleotides, each with a sugar-phosphate backbone and nitrogenous bases.

• Phosphodiester Bond: Formed between the phosphate group of one nucleotide and the sugar of the next.

• Hydrogen Bonds: Hold base pairs together (A-T: 2 bonds, G-C: 3 bonds).

Meselson-Stahl Experiment

This experiment demonstrated that DNA replication is semi-conservative: each new DNA molecule contains one old and one new strand.

• Conservative Model: Both strands remain together (disproved).

• Dispersive Model: DNA is a mix of old and new segments (disproved).

Introduction to DNA Replication

DNA replication is the process by which cells duplicate their DNA before cell division. It requires a suite of enzymes working together.

Origin of Replication & Replication Forks

Replication begins at specific DNA sequences called origins of replication (ORI). Prokaryotes typically have one ORI per chromosome, while eukaryotes have multiple ORIs.

• Replication Fork: The area where DNA is unwound and new strands are synthesized.

Unwinding the DNA: Topoisomerase, Helicase & SSBs

These proteins work together to unwind and stabilize DNA during replication.

• Topoisomerase: Relieves tension from supercoiling.

• Helicase: Separates the two DNA strands.

• Single-Stranded Binding Proteins: Prevent re-annealing of separated strands.

DNA Polymerases

DNA polymerases synthesize new DNA strands by adding nucleotides to a primer. In prokaryotes, DNA polymerase III is the main enzyme for elongation.

• Primase: Synthesizes RNA primer to start DNA synthesis.

• DNA Polymerase III: Extends the new DNA strand from the primer.

• DNA Polymerase I: Replaces RNA primers with DNA.

DNA Replication & Methylation

Methylation helps distinguish old DNA strands from new ones and plays a role in gene regulation and DNA repair.

• Methylation: Addition of a methyl group (–CH3) to DNA bases.

Leading & Lagging DNA Strands

During replication, the leading strand is synthesized continuously, while the lagging strand is synthesized in short fragments called Okazaki fragments.

• Leading Strand: Synthesized in the same direction as the replication fork movement.

• Lagging Strand: Synthesized in the opposite direction, requiring multiple primers.

Steps of DNA Replication

DNA replication involves several coordinated steps:

1. Topoisomerase relieves supercoiling.

2. Helicase unwinds the DNA.

3. Single-Stranded Binding Proteins stabilize the unwound DNA.

4. Primase adds RNA primers.

5. DNA Polymerase III synthesizes new DNA strands.

6. DNA Polymerase I replaces RNA primers with DNA.

7. DNA Ligase joins Okazaki fragments.

DNA Repair

DNA replication is not always perfect; errors can occur, but cells have proofreading and repair enzymes to correct mistakes.

• Proofreading: DNA polymerases have 3'→5' exonuclease activity to remove incorrect bases.

• Repair Enzymes: Fix mismatches and other errors.

Central Dogma of Molecular Biology

The central dogma describes the flow of genetic information: DNA → RNA → Protein.

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

• Translation: RNA is used to build proteins.

Introduction to Transcription

Transcription is the process of synthesizing RNA from a DNA template. Genes contain sequences that code for proteins.

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

• Terminator: DNA sequence where transcription ends.

Steps of Transcription

Transcription consists of three main steps:

1. Initiation: RNA polymerase binds to the promoter.

2. Elongation: RNA polymerase synthesizes RNA by adding nucleotides.

3. Termination: RNA polymerase releases the RNA transcript at the terminator.

Transcription Termination in Prokaryotes

Prokaryotes use two mechanisms for transcription termination:

• Rho-dependent termination: Rho protein causes RNA polymerase to dissociate from DNA.

• Rho-independent termination: Formation of a stem-loop structure in RNA causes dissociation.

Eukaryotic RNA Processing & Splicing

Eukaryotic mRNA undergoes modifications before translation:

• 5' Cap Addition: Protects mRNA and aids in ribosome binding.

• Poly-A Tail Addition: Stabilizes mRNA and regulates export.

• Splicing: Removal of non-coding introns; exons are joined to form mature mRNA.

Types of RNA

Cells use several types of RNA, each with specific functions:

• Messenger RNA (mRNA): Carries genetic code from DNA to ribosome.

• Ribosomal RNA (rRNA): Forms the core of ribosome structure and catalyzes protein synthesis.

• Transfer RNA (tRNA): Brings amino acids to the ribosome during translation.

Genetic Code

The genetic code is a set of rules that defines how the sequence of nucleotides in mRNA is translated into amino acids in a protein.

• Codon: A sequence of three nucleotides that specifies an amino acid.

• Redundancy: Multiple codons can code for the same amino acid.

Introduction to Translation

Translation is the process of building proteins using the encoded message of mRNA.

• Ribosome: The molecular machine that assembles proteins.

• tRNA: Delivers amino acids to the ribosome, matching codons with anticodons.

Ribosome Structure & Binding Sites

Ribosomes have three binding sites for tRNA:

• A site: Holds the incoming tRNA with its amino acid.

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

• E site: Releases the discharged tRNA.

Antibiotics & Ribosome Function

Many antibiotics target ribosomes to block protein synthesis in bacteria, exploiting differences between prokaryotic and eukaryotic ribosomes.

Key Equations & Concepts

• Base Pairing Rule: ,

• Central Dogma:

• Genetic Code:

Additional info: These notes include practice questions to reinforce key concepts and support exam preparation.