DNA is the code of life revision questions

Lecture 1: Introduction to the Structure and Function of DNA

1. Bacterial Transformation Detection

• Fred Griffith's Experiments:

  • Utilized Streptococcus pneumoniae.

  • Two Strains Analyzed:

    • Smooth (S) Strain:

      • Virulent; features a polysaccharide capsule preventing immune detection.

    • Rough (R) Strain:

      • Non-virulent; lacks capsule, easily destroyed by immune system.

  • Key Observations:

    • Individually, Live R strain and heat-treated S strain: no disease.

    • Combined: caused pneumonia, suggesting genetic material transfer.

  • Conclusion: Bacterial heritable changes indicate the process named transformation.

  • Later, Oswald Avery identified DNA as the heat-stable component responsible for transformation.

2. Suitability of Bacteriophages for Hershey and Chase Experiment

• Separation of DNA and Protein:

  • T2 Bacteriophages: Composed of DNA (inside) and protein (outer shell).

  • Radioactively labeled DNA (³²P) and protein (³⁵S) to track roles in infection.

• Efficient Host Infection:

  • Bacteriophages inject genetic material into bacteria, leaving protein outside.

  • Facilitated separation using a blender and centrifugation to observe genetic transfer.

  • Phages replicate quickly in host cells, allowing for fast observation of genetic effects.

3. Structure Determination of DNA

• Rosalind Franklin:

  • Used X-ray diffraction of DNA fibers to reveal helical structure.

• Watson & Crick:

  • Combined Franklin's data with chemical knowledge to develop the double-helix model.

  • Concluded DNA strands are antiparallel with specific base pairing through hydrogen bonds.

• Erwin Chargaff:

  • Showed base pair frequency rules: A=T and G=C (Chargaff’s rules).

4. DNA Replication Mechanism

• Predictions on replication mechanisms: Semi-conservative, conservative, dispersive.

• Meselson & Stahl Experiment:

  • Confirmed semi-conservative replication through DNA weight distribution patterns.

  • Resulting DNA consists of one original and one newly synthesized strand.

• Support from DNA Structure: Antiparallel double-helix structure implies semi-conservative replication.

Lecture 2: Fundamentals of DNA Replication

1. Biochemical Properties of DNA Polymerases

• Catalytic Activity: Catalyzes phosphodiester bond formation in 5' to 3' direction.

• Template-Dependent: Requires single-stranded templates.

• Primers: Needs RNA or DNA primers with free 3'-OH to initiate synthesis.

• Proofreading Ability: Exonuclease activity ensures high fidelity by removing mismatched nucleotides.

• Substrate Specificity: Primarily for dTNPs.

• Synthesis Rates: Varies (replicative polymerases fast; repair polymerases slower).

• Cofactors Required: Mg²⁺ or Mn²⁺ ions stabilize negative charges on phosphate backbone.

2. Additional Enzymes for DNA Synthesis

• DNA Helicase: Unwinds double strands to produce single-stranded DNA templates.

• Primase: Synthesizes short RNA primers to provide a starting point for DNA polymerase.

• Single-Stranded Binding Proteins (SSB): Stabilize single-stranded DNA.

• RNase H: Removes RNA primers post-synthesis.

• DNA Ligase: Joins fragments together.

• Telomerase (Eukaryotes Only): Extends telomeres to preserve genetic information.

3. Roles of DNA Polymerases Beyond Replication

• DNA Repair:

  • Example: Nucleotide excision repair where DNA Pol I fills gaps.

• DNA Recombination:

  • In homologous recombination, polymerase aids in repair synthesis and genetic exchange during meiosis.

4. Definitions

• Telomere: Repetitive DNA at the ends of chromosomes, protecting against degradation.

• Leading Strand: Synthesized continuously from a single primer (5' to 3').

• Lagging Strand: Synthesized discontinuously in Okazaki fragments from multiple primers.

• Helicase: Enzyme unwinding DNA in ATP-dependent process.

• Okazaki Fragment: Short DNA segments synthesized on the lagging strand.

• Primase: Synthesizes short RNA primers during replication.

5. The End Replication Problem

• Definition: DNA polymerase cannot replicate lagging strand ends, leading to unreplicated segments and potential genetic loss.

• Solution:

  • Telomerase adds repeats to the template strand allowing proper primer binding and preventing loss of genetic information post-division.

Lecture 3: Introduction to Bacterial Transcription (RNA Synthesis)

1. Differences Between RNA and DNA

• RNA is composed of ribose rather than deoxyribose.

• Uracil is present instead of thymine.

• Typically single-stranded.

2. Definitions

• Transcription Unit: Region of DNA copied during transcription; eukaryotes have single, prokaryotes can have multiple genes.

• Gene: Basic unit of genetic information encoding proteins.

• Promoter: DNA sequence for RNA polymerase binding to initiate transcription.

• Terminator: DNA sequence signaling the end of transcription.

3. Role of σ (Sigma) Factors in Transcription Initiation

• Recognizes promoters and binds RNA polymerase, unwinding DNA to start transcription.

• Binds at -10 and -35 promoter sites, aiding holoenzyme recruitment.

4. Mechanisms of Transcription Termination in Bacteria

• Intrinsic Terminators: RNA forms hairpin structures followed by uracils.

• Rho-Dependent Terminators: Requires Rho protein, an RNA helicase.

Lecture 4: Fundamentals of Translation (Protein Synthesis)

1. Evidence for Non-Overlapping Genetic Code

• Benzer’s Study on T4 Bacteriophage:

  • Point mutations altering only one amino acid indicate sequential codon reading.

• Brenner & Crick’s Frameshift Mutations:

  • Indicated codons operate in triplets.

2. Triplet Codon Assignment

• Nirenberg and Matthaei’s Poly-U Experiment:

  • Established that the code UUU encodes phenylalanine.

• Triplet Binding Assay: Used to assign codons to specific amino acids.

3. Constituents of Bacterial Ribosome

• Small Subunit (30S): Assembles with mRNA and tRNAs.

• Large Subunit (50S): Contains 3 tRNA binding sites: A, P, and E.

4. Additional Components for Protein Synthesis

• mRNA: provides information.

• Aminoacyl tRNA: Delivers amino acids to ribosome.

• GTP: Energy source for synthesis.

• Bacterial Proteins: Includes initiation, elongation, and termination factors.

5. Differences in Translation Between Eukaryotes and Bacteria

• Eukaryotic Ribosomes (80S) are larger than bacterial (70S).

• Transcription/Translation Location: Coupled in bacteria; separate compartments in eukaryotes.

• Polycistronic vs. Monocistronic: Bacteria can have multiple reading frames; eukaryotes generally have one per mRNA.

Lecture 5: Introduction to Gene Regulation in Bacteria

1. Comparison of Transcription, Replication, and Translation

2. Summary of Central Dogma

• Transcription converts DNA into RNA.

• DNA Replication copies DNA.

• Translation synthesizes proteins.

• Central dogma: DNA → RNA → Protein.

3. Transcription vs Translation Initiation

4. Lactose's Effect on the Lac Repressor

• Lac Operon: Control genes in lactose metabolism from a single promoter.

  • LacZ: Codes for β-galactosidase, breaking down lactose.

• Repression in Absence of Lactose: Lac repressor binds to operator, blocking transcription.

• Induction by Lactose Presence:

  • Lactose converted into Allolactose, which binds repressor, changing its shape and allowing transcription to proceed by displacing it from operator.

• Feedback Loop: Allolactose and β-galactosidase interact to regulate expression.

5. Role of RNA Secondary Structures in Bacterial Gene Expression Regulation

• Riboswitches: RNA elements in the 5' UTR of mRNAs regulating access to Ribosome Binding Sites (RBS).

  • Change shape in response to ligand binding, influencing translation.

• Attenuation Mechanism: Regulates based on amino acid availability, forming hairpin structures to halt or continue transcription.

Lecture 6: Function & Combinatorial Action of Transcription Factors

1. Binding of Transcription Factors to DNA

• Composed of distinct DNA-binding and activation domains.

• Binding domains recognize specific DNA sequences.

• Amino acid interactions with DNA induce stability and specificity.

2. Advantages of Multiple Transcription Factors

• Cooperative Control: Genes activated/repressed based on combinatory TF presence.

• Increased regulatory specificity, improved effect, and reduced off-target effects.

3. Mechanism of Transcription Activation

• Increases RNA polymerase stability at the promoter.

• Cooperative Binding: Enhances RNA polymerase engagement with the template, promoting transcription initiation.

4. Example of Positive Feedback in Transcription Control

• Bacteriophage λ Repressor: Functions as a switch through cooperative binding that regulates gene expression.

  • Promotes its own expression while repressing alternative pathways via intricate binding dynamics.

5. Example of Negative Feedback in Transcription Control

• At high λ repressor concentrations, it binds to low-affinity sites, inhibiting further expression.

Lecture 7: Mechanisms of Transcription and RNA Processing in Eukaryotes

1. Limited Post-Transcriptional Regulation in Bacteria

• No splicing, shorter mRNA half-life, and rapid growth necessitate efficient regulations often at transcription level.

2. Enhancers' Role in Gene Expression

• Enhance transcription by recruiting multiple TFs and allowing distant regulation.

3. Role of General Transcription Factors

• Critical for assembling RNA polymerase and facilitating transcription machinery.

• TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH constitute the general transcription machinery.

4. Splicing Steps

• Two-step transesterification reaction via the spliceosome for exon joining and intron removal.

  1. Adenine forms lariat structure.

  2. Exons ligated and introns degraded.

5. Splicing Regulation

• Mediated through regulatory RNA sequences and alternative splicing producing diverse mRNA isoforms.

6. Differences Between Eukaryotic and Bacterial Factors

• Eukaryotic GTFs consist of multi-protein complexes; bacterial sigma factors are singular.

7. Similarities Between Eukaryotic and Bacterial Factors

• Both play critical roles in promoter recognition and transcription initiation.

Lecture 8: Chromatin Structure and Gene Expression

1. Basic Composition of Nucleosome

• Octamer of histone proteins forming a core, packages ~150 bp of DNA with linker DNA.

2. Measuring Chromatin Accessibility

• Chromatin remodeling to assess interactions with transcription factors, using nuclease sensitivity for active promoter regions.

3. Histone Acetylation and Active Chromatin

• Facilitates opening of chromatin, enabling transcription factor access, and promoting known active chromatin states.

4. Targeting of Histone Modifications

• Via transcription factors that recruit chromatin modifiers for regulation.

5. Understanding “Spreading” of Chromatin

• Propagation of chromatin modifications can influence long-range gene expression through feedback and marks affecting neighboring nucleosomes.

6. DNA Methylation Distribution in Mammals

• Non-random patterns of methylation influencing gene regulation and chromatin stability, with varied methylation depending on genomic context and cellular state.

7. Effects of DNA Methylation on Transcription

• Generally inhibits transcription by hindering transcription factor binding, leading to chromatin compaction.