In-Depth Notes on DNA Function, Replication, and Gene Expression

HISTORY OF DNA DISCOVERIES

  • 1869 - Friedrich Miescher

    • Discovered DNA molecule in nuclei
    • Importance: Suggested involvement of DNA in inheritance.

  • 1928 - Fredrick Griffith

    • Transformed bacteria from nonvirulent to virulent strains.
    • Importance: Stated that some unknown factor must cause transformation.

  • 1944 - Oswald Avery

    • Confirmed Griffith’s work; determined transformation was caused by DNA.
    • Importance: Provided evidence for DNA as the material of inheritance.

  • 1950 - Erwin Chargoff

    • Discovered nucleotide ratios; Chargoff’s rule (A = T and G = C).
    • Importance: Showed that purines equal pyrimidines in DNA.

  • 1952 - Hershey & Chase

    • Conducted blender experiment; DNA carried into next generation.
    • Importance: Confirmed DNA is the material of inheritance.

  • 1951-1953 - Rosalind Franklin

    • Used X-ray crystallography to determine DNA’s helical shape.
    • Importance: Indicated DNA is a double helix.

  • 1953 - Watson & Crick

    • Created DNA model as a double helix with a sugar-phosphate backbone, identifying base pairs.
    • Importance: Fundamental understanding of DNA structure and function.

  • 1958 - Meselson & Stahl

    • Discovered semiconservative replication of DNA.
    • Importance: Demonstrated precision of DNA replication.

  • 1964 - Marshall Nirenburg

    • Elucidated how the genetic code works; developed the codon table.

STRUCTURE OF DNA

  1. Nucleotide Composition:

    • Composed of:
      • Phosphate group
      • 5-carbon sugar (deoxyribose)
      • Nitrogenous base (purines & pyrimidines)

  2. Nitrogenous Bases:

    • Purines: Double-ring structure (Adenine and Guanine).
    • Pyrimidines: Single-ring structure (Thymine and Cytosine).

  3. Double Helix Structure:

    • Sugar-Phosphate Backbone: Connected by phosphodiester bonds.
    • Base Pairing:
      • Chargaff’s rule supports A pairing with T and G pairing with C, connected by hydrogen bonds.
    • Antiparallel Strands:
      • One strand runs 3’ to 5’, the other 5’ to 3’.

  4. Semiconservative Replication:

    • Each daughter DNA contains one original and one new strand.
    • Occurs during S phase of interphase before cell division.

DNA REPLICATION

  1. Key Terms:

    • Origin of Replication:
      • Starting point of replication (1 in prokaryotes, multiple in eukaryotes).
    • Replication Fork:
      • The Y-shaped region where DNA strands are separated.
    • Leading Strand:
      • Synthesized continuously in the 3’ to 5’ direction.
    • Lagging Strand:
      • Synthesized discontinuously in the 5’ to 3’ direction using Okazaki fragments.

  2. Key Enzymes:

    • DNA Helicase: Unwinds the DNA double helix, forming the replication fork.
    • Topoisomerase: Alleviates torsional strain ahead of helicase.
    • RNA Primase: Synthesizes RNA primers for DNA polymerase.
    • DNA Polymerase III: Extends new DNA strands, synthesizing majority of DNA.
    • DNA Polymerase I: Replaces RNA primers with DNA nucleotides.
    • DNA Ligase: Seals gaps between Okazaki fragments.
    • Gyrase: Rewinds the DNA helix in daughter strands.

  3. Proofreading:

    • Over 130 repair enzymes, including specific DNA polymerases, help correct errors during and after replication.

  4. Chromosome Formation for Cell Division:

    • DNA wraps around histone proteins to form nucleosomes.

TELOMERES

  1. Function:

    • Repeating segments of non-coding DNA at the ends of chromosomes (telomeres) prevent degradation of chromosomes during replication.
    • Associated with cellular aging.

  2. Telomerase:

    • An enzyme that extends telomeres, active in germ cells.

PROKARYOTIC REPLICATION

  1. Characteristics:
    • Involves one circular strand of DNA with a single origin of replication.
    • Replication proceeds bidirectionally.
    • Terminus is found 180 degrees from the origin.

GENE EXPRESSION AND PROTEIN SYNTHESIS

  1. Function of Genes:

    • Each gene codes for one polypeptide.
    • Some proteins consist of multiple polypeptides.

  2. Types of RNA:

    • Ribosomal RNA (rRNA): Forms the structural component of ribosomes.
    • Messenger RNA (mRNA): Antiparallel copy of DNA, carries genetic information.
    • Transfer RNA (tRNA): Brings amino acids to the ribosome and has a corresponding anticodon region.

  3. Central Dogma of Biology:

    • DNA → RNA (transcription) → Proteins (translation).

EUKARYOTIC TRANSCRIPTION

  1. Location: Occurs in the nucleus.

  2. Phases of Transcription:

    • Initiation:
      • RNA polymerase binds to the promoter (TATA box).
    • Elongation:
      • RNA polymerase synthesizes pre-mRNA.
      • Uracil pairs with adenine instead of thymine.
    • Termination:
      • RNA polymerase recognizes the termination sequence and releases the pre-mRNA strand.

  3. Post-Transcription Modifications:

    • Addition of a modified guanine cap at the 5’ end.
    • Splicing by spliceosomes to remove introns and join exons.
    • Addition of a poly-A tail at the 3’ end.

PROKARYOTIC TRANSCRIPTION

  • Takes place in the nucleoid and directly after DNA replication, resulting in mRNA that is ready for translation.

TRANSLATION

  1. Location: Takes place at the ribosome in the cytoplasm.

  2. Components of Ribosomes:

    • Small subunit aligns with mRNA.
    • Large subunit houses codon and anticodon interaction.
    • A Site: Aminoacyl site, entry for new tRNA.
    • P Site: Peptidyl site, holds current tRNA and polypeptide.
    • E Site: Exit for tRNA.

  3. Steps of Translation:

    • Initiation: mRNA binds to the small subunit of ribosome.
    • Elongation:
      • New tRNA enters A site, peptide bond formed.
      • Translocation occurs as ribosome moves along mRNA.
    • Termination: Stop codon is reached, releasing polypeptide chain.
    • Polyribosomes: Multiple ribosomes can translate a single mRNA simultaneously.

THE GENETIC CODE

  • Codons (triplets of bases) specify amino acids.
  • Involves the "wobble effect": exact match not always needed for the third base.

REGULATION OF GENE EXPRESSION

  1. Prokaryotic Gene Regulation: Operon model regulates transcription.

    • Includes operons: Inducible (default off) and Repressible (default on).

  2. Eukaryotic Gene Regulation: Controlled at multiple levels;

    • Chromatin structure, transcriptional, post-transcriptional, translation, post-translational controls.

  3. Transcriptional Control: Involves transcription factors that bind RNA polymerase and assist transcription.

  4. Post-Transcriptional Control: Includes alternative RNA splicing and miRNA/siRNA effects on mRNA stability.

  5. Translational Control: Ability to block translation and determine mRNA lifespan.

  6. Post-Translational Control: Processing of proteins ultimately leads to their final active forms.

MUTATIONS

  1. Definition: Any change in the DNA sequence.
  2. Types of Mutations:
    • Point mutations: Affect one base pair (silent, missense, and nonsense mutations).
    • Frameshift mutations: Caused by insertions or deletions of bases, leading to altered translations.

BIOTECHNOLOGY

  1. Tools Used:

    • Recombinant DNA: Combining DNA from different sources using restriction enzymes.
    • Cloning: Generating multiple copies of a DNA sequence through vectors.
    • Polymerase Chain Reaction (PCR): Amplifies DNA samples using thermal cycles.
    • Gel Electrophoresis: Separates DNA fragments based on size.

  2. Applications of Biotechnology:

    • STR Analysis: Used in DNA fingerprinting for identification.
    • Genome Editing: CRISPR for targeted DNA alterations.
    • Transgenic Species (GMOs): Incorporation of foreign DNA for beneficial traits.
    • Gene Therapy: Correcting defective genes in patients.
    • Human Genome Project: Extensive sequencing initiative yielding insights into human genetics.

  3. Proteomics: Study of proteins in terms of structure, function, and interactions across different cellular contexts.