Chapter 10: DNA Structure and Analysis

Central Dogma of Molecular Biology

The central dogma describes the flow of genetic information within a biological system. It outlines how DNA is transcribed into RNA, which is then translated into protein.

• DNA → RNA → Protein: Genetic information is stored in DNA, transcribed into messenger RNA (mRNA), and then translated into proteins by ribosomes.

• Reverse Transcriptase: Some viruses (e.g., retroviruses) use reverse transcriptase to synthesize DNA from an RNA template.

• Example: HIV uses reverse transcriptase to integrate its genetic material into the host genome.

Experiments Proving DNA is the Genetic Material

• Griffith's Experiment (1928): Demonstrated transformation in Streptococcus pneumoniae — non-virulent bacteria became virulent when mixed with heat-killed virulent bacteria.

• Avery, MacLeod, and McCarty (1944): Identified DNA as the transforming principle by showing that only DNA could transfer virulence.

• Hershey & Chase (1952): Used bacteriophage T2 labeled with radioactive isotopes to show that DNA, not protein, enters bacterial cells and directs viral replication.

Nucleotide Structure

• Nucleotide: Consists of a phosphate group, a deoxyribose sugar, and a nitrogenous base (adenine, thymine, cytosine, or guanine).

• Phosphodiester Bonds: Link nucleotides together to form the DNA backbone.

DNA Structure

• Double Helix: DNA consists of two antiparallel strands (5'→3' and 3'→5').

• Complementary Base Pairing: Adenine pairs with thymine (A-T) via two hydrogen bonds; guanine pairs with cytosine (G-C) via three hydrogen bonds.

• Antiparallel Strands: The two DNA strands run in opposite directions.

Chargaff's Rules

• Base Pair Ratios: In DNA, the amount of adenine equals thymine, and the amount of guanine equals cytosine (A = T, G = C).

• Formula:

DNA vs. RNA

• DNA: Double-stranded, contains deoxyribose, bases are A, T, G, C.

• RNA: Single-stranded, contains ribose, bases are A, U, G, C.

Types of RNA

• mRNA (messenger RNA): Carries genetic code from DNA to ribosomes.

• tRNA (transfer RNA): Brings amino acids to ribosomes during translation.

• rRNA (ribosomal RNA): Structural and catalytic component of ribosomes.

• Regulatory RNAs: Includes small RNAs (e.g., siRNA, miRNA) that regulate gene expression.

DNA Melting and Hybridization

• Melting (Denaturation): Separation of DNA strands by breaking hydrogen bonds, usually by heat.

• Hybridization: Re-annealing of complementary DNA or RNA strands.

Electrophoresis

• Purpose: Separates DNA fragments by size using an electric field in a gel matrix.

• Smaller fragments: Move faster and farther through the gel.

Chapter 11: DNA Replication and Recombination

Semiconservative Replication

Each new DNA molecule consists of one parental and one newly synthesized strand.

• Meselson and Stahl Experiment: Used isotopic labeling to demonstrate semiconservative replication in E. coli.

DNA Polymerase and Synthesis Direction

• DNA Polymerase: Enzyme that synthesizes DNA in the 5′ → 3′ direction by adding nucleotides to the 3′ end.

• Leading Strand: Synthesized continuously toward the replication fork.

• Lagging Strand: Synthesized discontinuously away from the fork in short segments called Okazaki fragments.

Enzymes and Proteins in Replication

• Helicase: Unwinds the DNA double helix.

• Gyrase (Topoisomerase): Relieves supercoiling ahead of the replication fork.

• Single-Strand Binding (SSB) Proteins: Stabilize unwound DNA strands.

• Ligase: Joins Okazaki fragments on the lagging strand.

Origins of Replication

• Prokaryotes: Typically have a single origin of replication.

• Eukaryotes: Have multiple origins of replication per chromosome.

Telomeres and Telomerase

• Telomeres: Repetitive DNA sequences at chromosome ends that protect against degradation.

• Telomerase: An enzyme (a reverse transcriptase) that extends telomeres using an RNA template.

Chapter 13: The Genetic Code and Transcription

Genetic Code Properties

• Degenerate: Multiple codons can specify the same amino acid.

• Universal: The code is nearly universal among organisms.

• Triplet: Each codon consists of three nucleotides.

• Non-overlapping: Codons are read in sequence, without overlap.

Codons, Anticodons, and Wobble

• Codon: A sequence of three mRNA nucleotides that specifies an amino acid.

• Anticodon: A complementary three-nucleotide sequence on tRNA.

• Wobble: Flexibility in base pairing at the third codon position allows some tRNAs to recognize multiple codons.

Translation Initiation

• Start Codon: AUG codes for methionine and signals the start of translation.

Transcription

• Template Strand: The DNA strand used to synthesize RNA.

• Promoters: DNA sequences where RNA polymerase binds to initiate transcription.

• RNA Polymerase: Enzyme that synthesizes RNA from a DNA template.

Bacterial vs. Eukaryotic Transcription

• Bacteria: Transcription and translation are coupled; mRNA is often polycistronic.

• Eukaryotes: Transcription occurs in the nucleus; mRNA is monocistronic and undergoes processing.

mRNA Processing

• 5′ Cap: Modified guanine nucleotide added to the 5′ end.

• Poly-A Tail: String of adenine nucleotides added to the 3′ end.

• Splicing: Removal of introns and joining of exons by the spliceosome.

Introns vs. Exons

• Introns: Non-coding sequences removed from pre-mRNA.

• Exons: Coding sequences retained in mature mRNA.

Spliceosome Function

• Spliceosome: A complex of proteins and RNAs that catalyzes intron removal and exon ligation.

Nonsense Mutations

• Nonsense Mutation: A point mutation that introduces a premature stop codon, leading to truncated proteins.

Chapter 14: Translation and Proteins

Translation Machinery

• Ribosomes: Complexes of rRNA and proteins that catalyze protein synthesis.

• tRNA: Adaptor molecules that bring amino acids to the ribosome.

• Translation Factors: Proteins that assist in initiation, elongation, and termination of translation.

Genetic Code Calculations

• Number of Possible Codons: possible codons (since there are 4 nucleotides and codons are triplets).

Prokaryotic vs. Eukaryotic Translation

• Prokaryotes: Translation begins before transcription is complete; ribosomes bind to Shine-Dalgarno sequence.

• Eukaryotes: Translation occurs in the cytoplasm; ribosomes bind to the 5′ cap of mRNA.

Classic Genetics Experiments

• Beadle & Tatum: Demonstrated the "one gene–one enzyme" hypothesis using Neurospora crassa mutants.

Protein Structure-Function Relationships

• Primary Structure: Amino acid sequence.

• Secondary Structure: Alpha helices and beta sheets formed by hydrogen bonding.

• Tertiary Structure: Three-dimensional folding of a single polypeptide.

• Quaternary Structure: Association of multiple polypeptide chains.

Chapter 15: Gene Mutation, DNA Repair, and Transposition

Types of Mutations

• Missense Mutation: Alters a codon, resulting in a different amino acid.

• Nonsense Mutation: Converts a codon to a stop codon, truncating the protein.

• Frameshift Mutation: Insertion or deletion of nucleotides not in multiples of three, altering the reading frame.

• Silent Mutation: Alters a codon but does not change the amino acid due to code degeneracy.

Genetic Repair and Mutagenesis

• DNA Repair Mechanisms: Include direct repair, excision repair, mismatch repair, and recombinational repair.

• Mutagenesis: The process by which mutations are introduced, either spontaneously or by mutagens.

Heritable vs. Non-Heritable Mutations

• Heritable (Germline) Mutations: Occur in gametes and can be passed to offspring.

• Non-Heritable (Somatic) Mutations: Occur in body cells and are not inherited by offspring.

Chapter 16: Regulation of Gene Expression in Bacteria

Operon Concept

• Operon: A cluster of genes under the control of a single promoter and regulatory elements, transcribed as a unit.

• Example: The lac operon in E. coli controls lactose metabolism.

Negative vs. Positive Control

• Negative Control: Gene expression is inhibited by a repressor protein (e.g., lac repressor).

• Positive Control: Gene expression is activated by an activator protein (e.g., CAP-cAMP complex).

Constitutive Expression

• Constitutive Genes: Expressed continuously, regardless of environmental conditions.

lac Operon Regulation

• lacI: Encodes the lac repressor protein.

• lac Repressor Mutation: Can lead to constitutive expression of the operon.

• lacY: Encodes lactose permease, a transport protein for lactose uptake.

CAP Activation System

• Catabolite Activator Protein (CAP): Activates transcription of the lac operon in the absence of glucose.

• Glucose vs. Lactose: Glucose presence inhibits lac operon expression (catabolite repression).

trp Operon Attenuation

• Attenuation: A regulatory mechanism that reduces expression of the trp operon when tryptophan is abundant.

Small RNA Regulation

• Small RNAs (sRNAs): Regulate gene expression post-transcriptionally by base pairing with mRNAs.