DNA, DNA Replication, RNA SLIDES

DNA Structure & Replication Study Notes

Topic 1: Proving DNA is the Genetic Material

1. The Search for the Genetic Material

  • In the early 1900s, scientists struggled to identify which molecule contains the genes involved in inheritance.

  • In the mid-1800s, Gregor Mendel discovered that heritable factors were passed down from parents to offspring.

  • In 1910, Thomas Hunt Morgan proved that genes are carried on chromosomes, which serve as the mechanical mechanism of inheritance.

  • This narrowed down the identity of genetic material to two types of molecules found in chromosomes: proteins and DNA.

2. Griffith’s Experiment

  • Conducted in 1928 by Frederick Griffith with a pneumonia-causing bacterium.

  • Used two strains of bacteria:

    • Pathogenic (disease-causing) strain: Known as “S” (smooth), contains a sugar capsule that protects it from the immune system.

    • Nonpathogenic (harmless) strain: Known as “R” (rough), lacks a capsule.

  • Key Findings: Heat-killed pathogenic bacteria mixed with living nonpathogenic bacteria caused the living cells to become pathogenic. This phenomenon was termed "transformation," defined as a change in genotype and phenotype due to the assimilation of foreign DNA by a cell.

  • The composition of the transforming principle was unknown; it could have been carbohydrates, proteins, or nucleic acids.

3. Avery-McCarty-Macleod Experiment

  • In 1944, Avery, McCarty, and MacLeod demonstrated that DNA was the transforming principle from Griffith's experiment.

  • They isolated different solutions containing heat-killed S bacteria and tested which solutions caused transformation of the R strain bacteria.

  • Their findings indicated that only solutions with intact S strain DNA led to the transformation of R strain bacteria.

  • The scientific community remained skeptical, as many still believed proteins were the genetic material.

Experimental Design Details:

  1. Removed lipids and carbohydrates from heat-killed S cells, leaving proteins, RNA, and DNA.

  2. Subjected the solution to treatment with enzymes that destroy proteins, RNA, or DNA.

  3. Added R cells to each treatment to observe whether transformation had occurred:

    • If DNA is present: S cells appear, transformation occurs.

    • If DNA is absent: No S cells appear, no transformation occurs.

  • Conclusion: Transformation cannot occur without the presence of DNA, confirming DNA as the hereditary material.

4. Hershey-Chase Experiment

  • Conducted in 1952 by Alfred Hershey and Martha Chase, showing DNA as genetic material in the bacteriophage T2 (virus that infects bacteria).

  • T2 is nearly entirely composed of DNA and protein; upon infecting E. Coli, it reprograms the host cell to produce new viruses.

  • Method: Used radioactive labeling:

    • Batch 1: Sulfur-35 in phage protein.

    • Batch 2: Phosphorus-32 in phage DNA.

  • Results indicated that only the DNA entered the bacteria, confirming DNA as the molecule responsible for inheritance.

Topic 2: The Structure of DNA

1. Overview of DNA Structure

  • Known by the 1950s as a polymer of nucleotides, comprising a nitrogenous base, a five-carbon sugar, and a phosphate group. The precise 3-D structure remained unclear.

2. Nucleotides

  • A nucleotide consists of:

    • Phosphate group

    • Pentose (5-carbon) sugar: Deoxyribose in DNA.

    • The carbon atoms in the sugar are numbered from 1' (1 prime) to 5'.

    • Nitrogen-containing base bound to the 1' carbon, and the phosphate group bound to the 5' carbon.

3. Nitrogenous Bases

  • Divided into two families:

    • Pyrimidines (one ring): Cytosine (C), Thymine (T), Uracil (U).

    • Purines (two rings): Adenine (A), Guanine (G).

  • Thymine is exclusive to DNA, while Uracil is exclusive to RNA.

  • Mnemonic: “CUT Py” for pyrimidines (C, U, T); “Pure As Gold” for purines (A, G).

4. Polynucleotides

  • Nucleotides link via dehydration reactions to form a polynucleotide strand.

  • Covalent bonds form between the 3' carbon of one nucleotide and the phosphate group of the next, creating a phosphodiester linkage.

  • Polynucleotides have a 3' (hydroxyl) end and a 5' (phosphate) end; nucleotides are added during synthesis at the 3' end.

5. Franklin and Wilkins' Contribution

  • Rosalind Franklin used X-ray crystallography to deduce the 3-D structure of DNA, concluding two outer sugar-phosphate backbones and nitrogenous bases in the interior, suggesting a helical structure.

6. Watson and Crick’s Contribution

  • Watson and Crick determined DNA's structure based on Franklin’s images, concluding:

    • DNA is helical and features two strands forming a double helix.

    • Width and base-spacing indicated a complementary base pairing where A pairs with T, and G pairs with C.

  • Chargaff’s rule established that A equals T, and C equals G in organisms, demonstrating the variability in base percentage among species.

7. 3D Structure of DNA

  • The DNA is a double-stranded molecule with alternating sugar-phosphate backbones, twisting to form a helix held by hydrogen bonds between nitrogenous bases:

    • Cytosine pairs with Guanine (three hydrogen bonds).

    • Adenine pairs with Thymine in DNA (two hydrogen bonds) or Uracil in RNA.

  • The strands are antiparallel, one running 5'→3' and the other 3'→5'.

8. DNA in Prokaryotes vs Eukaryotes

  • Prokaryotic DNA forms a single circular chromosome, while eukaryotic DNA organizes into linear chromosomes.

  • Bacteria also possess plasmids, small circular DNA molecules granting genetic advantages like antibiotic resistance.

Topic 3: The Replication of DNA

1. Basic Principle of DNA Replication

  • Watson and Crick’s model proposed that each strand serves as a template for new strand synthesis during replication, unwinding the parent molecule.

2. Semiconservative Model

  • The semiconservative model stipulates that each daughter strand consists of one original and one newly formed strand. Proven by Matthew Meselson and Franklin Stahl in the late 1950s, it contrasted with conservative and dispersive models.

3. Meselson-Stahl Experiment

  • DNA was labeled with heavy isotope nitrogen (15N), then transferred to normal nitrogen (14N) for replication.

  • Results showed:

    • After one cycle, all DNA had a mix of heavy and light isotopes.

    • After two cycles, half contained only light and half retained a mix. This provided evidence for the semiconservative model.

4. Origins of Replication

  • Replication begins at specific origins of replication (ori) creating replication bubbles.

  • Eukaryotic chromosomes can have numerous origins, with replication forks representing new strand elongation points.

5. DNA Replication Complex

  • Involves multiple proteins, forming a large complex with key players including:

    • DNA Polymerases: Enzymes that synthesize new DNA.

    • Primase: Enzyme creating RNA primers.

    • DNA Ligase: Joins Okazaki fragments.

    • Helicase: Unwinds DNA double helix.

    • Topoisomerase: Alleviates twisting strain ahead of the replication fork.

6. Unwinding the Double Helix

  • Helicase unwinds the double helix at replication forks, separating strands.

  • Topoisomerase resolves strain caused by unwinding.

7. DNA Polymerase and Directionality

  • DNA polymerases add nucleotides to elongate new strands at the replication fork, only in the 5'→3' direction.

  • They read the template strand from 3'→5', translating into a 5'→3' complementary strand.

8. Leading and Lagging Strands

  • Due to directional synthesis, one strand (leading) can be synthesized continuously towards the replication fork, while the lagging strand is synthesized in segments (Okazaki fragments) moving away from the fork, ultimately joined by DNA ligase.

9. Priming DNA Synthesis

  • DNA polymerases cannot initiate synthesis; replication starts with a short RNA primer synthesized by primase.

  • For the leading strand, one primer suffices, while each lagging fragment requires a new primer which later gets replaced with DNA.

10. Proofreading and Repairing DNA

  • DNA polymerases can rectify mistakes during replication via proofreading and mismatched repair processes, which ensure base pairing fidelity.

Topic 4: The Structure & Function of RNA

1. The Structure of RNA

  • RNA consists of nucleotide monomers comprising sugar, phosphate, and a nitrogenous base, forming a linear molecule with ends marked as 3' and 5'.

  • Differences from DNA:

    • RNA is usually single-stranded and contains ribose (instead of deoxyribose).

2. Nitrogenous Bases in RNA

  • RNA nitrogenous bases include pyrimidines (C, U) and purines (A, G).

  • RNA exhibits complementary base pairing:

    • Cytosine pairs with Guanine (C-G).

    • Adenine pairs with Uracil (A-U).

3. Functions of RNA

  • Diverse roles of RNA include:

    • mRNA: Messenger that carries genetic info.

    • rRNA: Component of ribosomes, critical for protein synthesis.

    • tRNA: Transports amino acids to ribosomes for polypeptide assembly.

    • miRNA: Regulates gene expression.

    • Ribozymes: RNA molecules that catalyze biochemical reactions.

4. The 3 Main Types of RNA

  • mRNA: Transfers genetic code from DNA to ribosomes; codons (3 nucleotides) specify amino acids.

  • rRNA: Associates with proteins to form ribosomes, integral to protein synthesis.

  • tRNA: Delivers specific amino acids to ribosomes based on codon-anticodon matching.

5. Self-Assessment Questions Overview

  • Various self-assessment questions highlight understanding of DNA and RNA structure, replication processes, and functional implications. Use these to practice and gauge comprehension of material.

6. Answers to Self-Assessment Questions

  • Answers provided for review and self-testing purposes, reinforcing and assessing mastery of topics pertaining to DNA structure and replication processes, RNA functions, and genetic material concepts.