DNA Structure, Function, and Gene Expression

Historical Foundations of DNA Discovery

• 1863: Gregor Mendel

  • A monk known as the father of genetics.

  • Conducted experiments with peapods to explain patterns of inheritance.

  • Played a critical role in the discovery of genes and heredity.

• 1869: Johann Friedrich Miescher

  • A Swiss physician who first isolated DNA.

  • Discovered a microscopic substance in the nuclei of white blood cells from pus on discarded surgical bandages.

  • Initially named the substance "nuclein."

  • Determined that nuclein was not a protein and was rich in nitrogen and phosphorus ($P$).

• 1878: Albrecht Kossel

  • Isolated the non-protein component of nuclein, identifying it as nucleic acid.

  • Subsequently isolated the five primary nucleobases.

• 1919: Phoebus Levene

  • Identified the constituent base, sugar, and phosphate units that form a nucleotide.

  • Suggested that DNA consisted of a string of nucleotide units linked through phosphate groups.

• 1928: Frederick Griffith

  • Investigated a vaccine against Streptococcus pneumoniae.

  • Conducted four experiments injecting different bacterial strands into mice, leading to a breakthrough in understanding heredity.

• 1937: William Astbury

  • Produced the first X-ray diffraction patterns demonstrating that DNA possessed a regular structure.

• 1944: Avery-MacLeod-McCarty Experiment

  • Demonstrated definitively that isolated DNA was the material making up genes and chromosomes.

• 1950: Erwin Chargaff

  • Discovered two rules regarding base pair makeup that hinted at the double helix structure.

  • Chargaff's rules state that the ratio of Adenine ($A$) to Thymine ($T$) and Guanine ($G$) to Cytosine ($C$) is always close to unity ($1:1$).

• 1952: Maurice Wilkins and Rosalind Franklin

  • Franklin, a British biophysicist and X-ray crystallographer at King's College, generated key data.

  • Obtained high-quality X-ray diffraction images of DNA fibers, showing a scattering pattern that translated into a 3D shape.

• 1953: James Watson and Francis Crick

  • Examined the DNA structure using previous X-ray diffraction photos.

  • Discovered the characteristic "X" shape indicating a helical structure.

  • Formally proposed the double helix model in the journal Nature (April 25, 1953).

• Post-Discovery Milestones

  • 1962: Watson, Crick, and Wilkins awarded the Nobel Prize.

  • 1977: Sanger and colleagues introduced the "Sanger method" for rapid and accurate DNA sequencing.

  • 1977-1979: Genentech used genetic engineering to produce human somatostatin, insulin, and growth hormone.

  • 1983: Kary Mullis published the first paper describing Polymerase Chain Reaction ($PCR$).

  • 1984: Planning began for the Human Genome Project ($HGP$), funded by the US government.

  • 1990: The first FDA-approved gene therapy experiment occurred in the US; the HGP officially began.

  • 1996: Dolly the Sheep became the first mammal cloned from an adult animal cell.

  • 2003: Completion of the Human Genome Project announced with $99.99\%$ accuracy.

The Chemical and Molecular Structure of DNA

• Definition and Function

  • DNA (Deoxyribonucleic Acid) is the "blueprint for life."

  • It encodes genetic instructions for development and functioning in living organisms and viruses.

  • It determines physical characteristics (e.g., hair/eye color) and disease predisposition.

  • Length context: The total length of DNA in the human body is equivalent to $120 \text{ billion miles}$.

• The Nucleotide Monomer

  • DNA is a nucleic acid composed of four types of nucleotides.

  • Each nucleotide consists of:

  1. Phosphate group: Attached to the $5'$ carbon of the sugar.

  2. Sugar (Deoxyribose): A five-carbon sugar.

  3. Nitrogenous base: Attached to the $1'$ carbon.

• Nitrogenous Bases

  • Pyrimidines (Single-ring structures):

  • Thymine ($T$): Includes a methyl group ($H_3C$).

  • Cytosine ($C$).

  • Purines (Double-ring structures):

  • Adenine ($A$).

  • Guanine ($G$).

• The Polynucleotide Chain

  • Nucleotides are joined by phosphodiester linkages.

  • This linkage is formed via a condensation reaction between the $3'$ hydroxyl ($OH$) group of one sugar and the phosphate group on the $5'$ carbon of the next sugar, releasing $H_2O$.

  • This forms the "sugar-phosphate backbone."

• The Watson-Crick Double Helix Model

  • Described as a "twisted rope-ladder."

  • Sides: Represent the sugar-phosphate backbones.

  • Rungs: Represent pairs of nitrogenous bases connected by hydrogen bonds.

  • Antiparallel Orientation: The two strands run in opposite directions. One strand runs $5' \rightarrow 3'$ while the other runs $3' \rightarrow 5'$.

  • Complementary Base Pairing:

  • Adenine pairs with Thymine ($A-T$).

  • Guanine pairs with Cytosine ($G-C$).

  • Bonding requires one purine and one pyrimidine.

  • Structural Parameters:

  • Vertical residue distance: $3.4 \text{ Å}$.

  • Repeat unit: Every $10$ residues or $34 \text{ Å}$.

  • Angle between residues: $36^{\circ}$.

  • Distance of phosphorus to axis: $10 \text{ Å}$.

The Mechanism of DNA Replication

• Process Overview

  • Occurs in the nucleus during the $S$ phase before cell division.

  • Follows a semi-conservative model: Each new double helix consists of one parental (old) strand and one daughter (new) strand.

• Steps of Replication

  • Unwinding: The DNA helicase enzyme separates the two parental strands.

  • Priming: Primase prepares the site for synthesis.

  • Synthesis: DNA Polymerase adds nucleotides to the template strands.

• Directionality and Enzymes

  • DNA Polymerase only adds nucleotides to the $3'$ end of a growing strand.

  • DNA synthesis always moves in the $5' \rightarrow 3'$ direction.

  • Leading Strand: Constructed continuously in the direction of the replication fork.

  • Lagging Strand: Constructed in segments called Okazaki fragments (named after the discovering scientists).

  • DNA Ligase: Joins Okazaki fragments together where they meet to form a continuous strand.

• Fidelity

  • DNA Polymerase has a proofreading function to fix errors.

  • Replication errors (wrong base, deletion, or addition) that remain unfixed result in mutations, which are permanent changes in the DNA sequence.

Gene Expression: Transcription

• Central Dogma

  • The molecular "chain of command" is: DNA $\rightarrow$ RNA $\rightarrow$ Protein.

  • DNA remains in the nucleus; proteins are synthesized in the cytoplasm.

  • RNA acts as the bridge.

• Transcription Process (Nucleus)

  • RNA Polymerase: The primary enzyme involved.

  • Promoter: A specific DNA sequence where RNA polymerase attaches to initiate transcription.

  • Template: Only one DNA strand serves as the template. The DNA rewinds after the polymerase passes.

  • Termination: Transcription stops when the polymerase reaches the end of the gene marked by a specific sequence.

• Post-Transcriptional Modifications (Eukaryotes)

  • Before leaving the nucleus, the pre-mRNA (RNA transcript) undergoes processing:

  1. $5'$ Cap: Addition of a modified guanine cap.

  2. $3'$ Poly-A tail: Addition of a long chain of adenine nucleotides.

  3. Splicing: Introns (non-coding regions) are removed, and Exons (coding sequences) are spliced together.

The Genetic Code and RNA Functional Roles

• Types of RNA

  • Messenger RNA (mRNA): Carries the protein-building message; specifies amino acid sequence.

  • Transfer RNA (tRNA): Delivers specific amino acids to the ribosome during translation; contains an anticodon that matches the mRNA codon.

  • Ribosomal RNA (rRNA): A component of ribosomes along with proteins.

• The Genetic Code

  • Based on a triplet code: Three RNA bases constitute a codon.

  • There are $4^3 = 64$ possible codons but only $20$ amino acids.

  • Start Codon: $AUG$ (codes for Methionine, or Met).

  • Stop Codons: $UAA, UAG, UGA$ (do not code for amino acids).

  • Redundancy: Multiple codons can code for the same amino acid (e.g., $GAC$ and $GAU$ both code for Aspartic Acid).

  • Universality: The code is nearly the same for all living organisms.

Protein Synthesis: Translation

• Translation Mechanism (Cytoplasm)

  • The ribosome (composed of large and small subunits) interacts with mRNA and tRNA to build a polypeptide.

• Steps of Translation

  • Initiation: The small ribosomal subunit binds to mRNA. An initiator tRNA (carrying Met) binds to the start codon at the P site.

  • Elongation:

  1. Codon Recognition: A new tRNA enters the A site.

  2. Peptide Bond Formation: The ribosome catalyzes a bond between the growing polypeptide and the new amino acid.

  3. Translocation: The ribosome moves along the mRNA, shifting the tRNA from the A site to the P site.

  • Termination: A stop codon enters the A site. The completed polypeptide is freed, and the ribosome subunits dissociate.

Mutations and Genetic Variation

• Sources of Mutation

  • Spontaneous: Random errors during replication or recombination.

  • Mutagens: Physical or chemical agents (e.g., X-rays, UV light).

• Types of Point Mutations

  • Base-pair Substitution: Replacement of one nucleotide with another.

  • Silent: No change in the amino acid sequence.

  • Missense: Changes one amino acid to another (e.g., Serine instead of Glycine).

  • Nonsense: Changes a codon into a stop codon, likely resulting in a nonfunctional protein.

  • Frameshift Mutations: Result from the Insertion or Deletion of nucleotides.

  • These disrupt the entire reading frame from the point of mutation onward, usually resulting in completely nonfunctional polypeptides.

• Case Study: Sickle Cell Anemia

  • Caused by a base-pair substitution in the beta globin gene.

  • Thymine ($T$) is replaced by Adenine ($A$) in DNA, leading to a change in mRNA.

  • The sixth amino acid changes from Glutamic acid (Glu) to Valine (Val).

  • Results in HbS (sickle hemoglobin), causing red blood cells to clump and form a crescent shape.

Questions & Discussion

• Q: Name the two scientists who described the structure of the DNA molecule?

  • A: James Watson and Francis Crick.

• Q: The work of __________________ helped the above scientists with their description of the DNA molecule.

  • A: Rosalind Franklin (and Maurice Wilkins).

• Q: The work of Hershey and Chase determined that ____________ is the genetic material.

  • A: DNA.

• Q: The monomers of nucleic acids are called ______________.

  • A: Nucleotides.

• Q: The functional group at the 5’ end of a nucleotide strand is _____________ whereas the functional group at the 3’ end is _________.

  • A: Phosphate; Hydroxyl ($OH$).

• Q: What are the main enzymes involved in DNA replication?

  • A: Helicase, Primase, DNA Polymerase, and DNA Ligase.

• Q: In what direction does DNA Polymerase work?

  • A: $5' \rightarrow 3'$.

• Q: One strand of DNA is copied ____________ while the other strand is copied in ________. Called ______________.

  • A: Continuously; segments; Okazaki fragments.

• Q: What is transcription and where does it take place?

  • A: The process of making RNA from a DNA template; takes place in the nucleus.

• Q: What is the main enzyme involved in transcription?

  • A: RNA Polymerase.

• Q: List the three types of RNA molecules?

  • A: mRNA, tRNA, rRNA.

• Q: What is mRNA processing?

  • A: The addition of a $5'$ cap, a $3'$ poly-A tail, and splicing (removal of introns).

• Q: Where does translation take place?

  • A: In the cytoplasm (on ribosomes).

• Q: In translation, the starting molecule is _____________ and the resulting molecule is ______.

  • A: mRNA; a polypeptide (protein).

• Q: Base substitution can result in three types of mutation. List them:

  • A: Silent, Missense, and Nonsense.

• Q: Frameshift mutations result from ___________ or _____________ of a base.

  • A: Insertion; Deletion.