PYTR Class

Learning Objectives

1. [AHL] Explain the directionality of RNA and DNA.
2. [AHL] Describe the role of purine-to-pyrimidine bonding in maintaining DNA helix stability.
3. [AHL] Identify the structure and function of a nucleosome.
4. [AHL] Analyse the evidence from the Hershey-Chase experiment supporting DNA as the genetic material.
5. [AHL] Interpret Chargaff’s data on the relative amounts of pyrimidine and purine bases across diverse life forms.

Part 1: Directionality of RNA and DNA

Directionality of RNA and DNA in Enzymatic Processes

• Directionality affects key biological processes, ensuring correct molecular interactions:
  • Replication – DNA polymerases copy DNA.
  • Transcription – RNA polymerase creates an RNA copy of DNA.
  • Translation – Ribosomes read RNA to determine the amino acid sequence of a polypeptide.
• Correct orientation is essential for DNA and RNA strands to fit into the active sites of enzymes and ribozymes.

Replication

• DNA nucleotides are added to the 3′ end of the growing strand.
• The 5′ phosphate of a free nucleotide binds to the 3′ deoxyribose sugar of the existing strand.
• DNA replication occurs in the 5′ to 3′ direction.
• Both DNA strands serve as templates, but since they are antiparallel:
  • One strand is synthesized in the same direction as replication.
  • The other strand is synthesised in the opposite direction, leading to differences in the process.

Transcription

• RNA nucleotides are added to the 3′ end of the growing RNA strand.
• The 5′ phosphate of a free nucleotide links to the 3′ ribose sugar of the existing strand.
• Transcription also occurs in the 5′ to 3′ direction.
• Only one of the two DNA strands is used as a template for RNA synthesis.
• The RNA strand is assembled in the same direction as transcription proceeds.

Translation

• RNA carries the genetic instructions for building a polypeptide.
• The ribosome moves along the RNA strand from 5′ to 3′, linking amino acids in sequence.
• Translation follows the 5′ to 3′ direction just like replication and transcription.

Part 2: Maintaining DNA Helical Structure

Purine-pyrimidine

• Nitrogenous bases in DNA are classified into two groups:
  • Purines
    • Adenine (A) and Guanine (G)
    • Have two rings of atoms.
  • Pyrimidines
    • Cytosine (C) and Thymine (T)
    • Have one ring of atoms.
• Base pairing in DNA follows a strict purine-to-pyrimidine rule:
  • For each base pair, if one is a purine and one must be complimented with a pyrimidine.
• This ensures equal width for all base pairs, maintaining a uniform structure in the double helix.
• The consistent spacing stabilises the DNA molecule and allows for any base sequence in genes.

Chargaff’s rules

• Base Pairing Rule
  • Amount of A = amount of T and,
  • Amount of C = amount of G
  • This provided evidence for complementary base pairing in DNA.
• Purine-Pyrimidine Balance
  • A and G are purines, and C and T are pyrimidines
  • Chargaff’s data showed that:
    • Total purine content = total pyrimidine content in a DNA sample.
• Species Variation
  • A = T and C = G ratios remained constant within a species
  • The proportions of bases varied between species.
  • This suggested that DNA composition is unique to each organism, supporting its role as the genetic material.

Part 3: What are Nucleosomes?

• Eukaryotes (plants, animals, and other eukaryotic organisms) have nucleosomes, where DNA is wrapped around histone proteins.
• Bacteria lack nuclei and histones, meaning their DNA is “naked” and not associated with proteins for packaging.

Chromosomes as Condensed DNA

• Chromosomes are highly condensed forms of DNA, allowing genetic material to fit within the cell nucleus.
• DNA condenses by wrapping around histone proteins, forming nucleosomes, which further coil and fold into chromatin.
• During cell division (mitosis and meiosis), chromatin condenses even further to form distinct chromosomes, making genetic material easier to separate.
• Chromosome structure ensures accurate DNA replication and distribution to daughter cells.

Steps in DNA Condensation

1. Nucleosomes formation
   • DNA stand wraps histone octamer and reinforced by H1 protein
2. Solenoid formation
   • The nucleosomes coil to form a loop or a spring structure
3. Chromatin fibre formation
   • Solenoid fibre further condense by coiling and looping about a scaffolding protein
   • Two types of chromatin:
     • Euchromatin: Loosely packed – available for gene expression i.e transcriptionally active.
     • Heterochromatin: Densely packed – unavailable for gene expression i.e transcriptionally inactive.

Part 4: Evidence for DNA as Genetic Material

Hershey–Chase Experiment: Evidence for DNA as Genetic Material

• Scientists knew chromosomes played a role in heredity but were unsure if DNA or protein was the genetic material.
• Protein was initially considered the better candidate due to its complexity (20 amino acids vs. 4 nucleotide types in DNA).

• Method:
  1. Viruses with labeled protein or DNA infected bacteria.
  2. A blender separated viral coats from bacteria.
  3. Centrifugation concentrated bacteria into a pellet.
  4. Radioactivity was measured in the pellet (bacteria) and supernatant (viral coat).

• Results & Conclusion:
  • Radioactive phosphorus (32P) was found inside the bacteria, proving DNA entered the cells.
  • Radioactive sulfur (35S) remained in the viral coat, meaning protein did not enter the cells.
  • Conclusion: DNA, not protein, is the genetic material responsible for viral replication.

Questions

1. Differentiate between a supernatant and a pellet. [2]
2. Justify why the genetic material should be located in the pellet rather than the supernatant. [2]
3. Identify the percentage of ³²P that remains in the supernatant. [1]
4. Calculate the percentage of ³²P that is found in the pellet after centrifugation. [2]
5. Evaluate the evidence supporting DNA as the molecule responsible for transforming bacteria into infected cells. [3]