AP Biology Unit 6: Gene Expression

Central Dogma of Molecular Biology

DNA makes RNA makes proteins aka DNA triplets → mRNA codons → amino acid sequence (protein).

DNA

• Double stranded helical molecule made of nucleotide monomers, with each nucleotide consisting of:

  • deoxyribose sugar (5 carbon sugar)

  • phosphate group

  • 1 of 4 nitrogenous bases (A, T, C, or G)

• Sugar and phosphate form sugar phosphate backbone connected by covalent bonds

• Nitrogenous bases pair the helical structure through hydrogen bonds because of the complementary base pairing (AT CG)

• Strands are anti-parallel: one runs 5’ to 3’ and another 3’ to 5’ for base pairing to occur

4 Characteristics that make DNA’s life primary genetic molecule

1. Information Storage:

   • DNA’s sequence of bases (e.g., ACGT) acts as an informational code specifying RNA and protein sequences.

2. Replicability:

   • Base-pairing (A-T, G-C) allows DNA strands to serve as templates for complementary strand synthesis, ensuring accurate replication.

3. Stability:

   • The double helix protects the bases, making DNA a stable molecule.

4. Mutability:

   • DNA can mutate (spontaneously or due to environmental factors), allowing for genetic variation and evolution.

RNA

• Single stranded molecule involved in protein synthesis and gene regulation

  • 3 types of RNA:

    • mRNA: messenger RNA

    • tRNA: transfer RNA

    • rRNA: ribosomal RNA

• Bases are AU and CG

• Sugar is ribose

Prokaryotic vs. Eukaryotic Genetic Information Storage

• Prokaryotes:

  • DNA is stored in looped circular chromosomes (no start or end).

  • Genomes are small (100,000 to 10 million base pairs) and naked (not wrapped around proteins).

• Eukaryotes:

  • DNA is stored in linear chromosomes.

  • DNA is wrapped around proteins called histones.

  • Eukaryotic genomes are much larger than prokaryotic genomes.

Plasmids

• Small, extra-chromosomal loops of DNA found in bacteria that facilitate:

  • horizontal gene transfer (transferring antibiotic resistant genes)

  • genetic engineering for DNA replication and expressing engineered genes in bacteria

  • bacterial conjugation: transfer of plasmids between cells

Origin of Replication

• Specific sequence where replication begins

Helicase

• Unzips DNA via breaking hydrogen bonds between strands creating a replication fork

DNA Polymerase

• Synthesizes new strands by adding nucleotides to the 3’ end of a growing (extends from existing) strand

Single-Strand Binding Proteins

• Prevent re-winding of DNA strand

Primase

• Lays down RNA primers to provide a starting point for DNA polymerase

Ligase

• Seals gaps between fragments

Topoisomerase

• Relieves supercoiling during DNA replication via cutting & rejoining DNA strands

Leading Strand

• Synthesized continuously in same direction as replication fork opens

• Differs from lagging strand b/c of directionality of DNA polymerase

Lagging Strand

• Synthesis moves in opposite direction of the opening replication fork by forming Okazaki fragments each requiring a new primer

DNA Replication

• Semiconservative process with each strand of the double helix acts as a template for synthesizing a new complementary strand

• Process:

  • Enzymes pull the double helix apart.

  • Each single strand acts as a template.

  • Free nucleotides in the nucleus pair with exposed bases following base-pairing rules:

    • A pairs with T, and C pairs with G.

Transcription

Process in the nucleus

1. Begins at the promoter region on the DNA, marking the gene’s starting point

2. RNA polymerase binds to the promoter and synthesizes RNA based on the DNA template strand

3. DNA is read in 3’ to 5’ direction and RNA is synthesized in the 5’ to 3’ direction

4. When RNA polymerase reaches a terminator region, transcription ends

DNA Template Strand

• DNA strand that’s transcribed into RNA

Coding Strand

• Complementary to the template strand that shares the same sequence as mRNA except RNA has U instead of T

Unique Features of Prokaryotic Transcription

• Prokaryotes lack a nucleus so transcription and translation occur simultaneously in the cytoplasm

• Transcribed RNA can be immediately translated by ribosomes

• Polysomes: Multiple ribosomes can translate the same RNA strand simultaneously

Genetic Code

• The code used by living organisms to translate nucleotide sequences (RNA) into amino acid sequences (proteins).

How to read the genetic code

1. Use a genetic code chart (which can be in a circular or tabular format)

2. Match the first, second, and third bases of a codon to determine the amino acid it codes for.

   • Example: AUG codes for methionine (met); GAA codes for glutamic acid (Glu

Translation

• Occurs in the cytoplasm

• Converts an mRNA sequence into a polypeptide to produce a specific sequence of amino acids that form a polypeptide which then folds to become a functioning protein involving 3 key players:

  • mRNA: contains codons specifying order of amino acids

  • tRNA: transfers specific amino acids to the ribosome that consists of:

    • an anticodon that pairs w/ the mRNA codon

    • amino acid binding site that carries the amino acid

  • ribosome: synthesizes the polypeptide by forming peptide bonds between amino acids

Ribosome

• Consists of a large subunit and small subunit that comes together to form a ribosome found in the cytoplasm or rough ER

• Has three tRNA binding sites:

  • A site: accepts new tRNA with an amino acid

  • P site: Holds the growing polypeptide

  • E site: Where tRNA exits after giving up its amino acid

Steps of Translation

1. Inititiation:

   1. mRNA leaves the nucleus and attatches to the small ribosomal unit, identifying its start codon AUG. Large ribosomal subunit idenfies UAC (complementary codon) and joins to the small unit, starting translation

2. Elongation:

   1. tRNA brings amino acids to the ribosome that forms peptide bonds between amino acids in P and A site. Ribosomes move the tRNA to the E site to exit and repeat the process as the polypeptide chain forms and grows

3. Termination:

   1. Ribosomes reach a stop codon and a release factor binds to the ribosome to release the polypeptide that folds into a functional protein

Operons

• A cluster of genes in bacteria that are controlled together and transcribed into a single mRNA, allowing the cell to turn multiple genes on or off at the same time.

Trp Operon (Repressible Operon)

• Makes tryptophan (Amino acid) only when the cell doesn’t have enough

• How it works:

  • When tryptophan is LOW (absent) => ON

    • Repressor is inactive (can’t bind to operator)

    • RNA polymerase makes tryptophan

    • Tryptophan production eventually represses its own synthesis (negative feedback).

  • When tryptophan is HIGH (present) => OFF

    • Tryptophan acts as a repressor by binding to the operator, blocking transcription

Repressible Operon

• Default condition is “on”

• Presence of tryptophan turns system off

Lac Operon

• Breaks down lactose when it’s available

• How it works:

  • When lactose is ABSENT → OFF

    • Repressor is active

    • Binds to operator → blocks transcription

  • When lactose is PRESENT → ON

    • Lactose binds to the repressor that become inactive (can’t bind operator)

    • RNA polymerase creates enzymes to break lactose

Inducible Operon

• Default condition is “off”

• Lactose turns the system on as the ‘inducer’

Glucose vs. Lactose Metabolism (AKA: “Diauxic” or “Two-Phase” Growth)

• When provided with glucose and lactose:

  • E. coli prefers glucose as it’s easier to metabolize (monosaccharide vs. disaccharide)

  • When glucose runs out, there’s a lag while the lac operon activates and produces enzymes to digest lactose.

Euchromatin

• Loosely packed (often acetylated) DNA that is transcribed into RNA and translated into protein.

Heterochromatin

• tightly packed (often methylated) DNA that is NOT transcribed

RNA Processing in Eukaryotes *Why it’s more complex*

1. Introns and Exons

   1. Introns: non-coding sequences removed during RNA splicing

   2. Exons: Coding sequences spliced together to form mRNA for translation

2. Post-Transcriptional Modifications in mRNA

   1. 5’ GTP Cap:

      1. Protects mRNA from degradation

      2. Aids in nuclear export and ribosome binding

   2. 3’ Poly-A Tail:

      1. Enhances mRNA stability and delays breakdown

microRNAS

• Regulates gene expression by forming RNA silencing complexes that either pause translation or degrade mRNA so that it can no longer be translated

Mutation

• A random change in DNA or an entire chromosome

Point Mutation

• A change in a single nucleotide (ex: C to T)

  • Types of point mutations:

    • Silent Mutation: DNA changes, but the amino acid and protein remain the same due to redundancy in the genetic code.

    • Nonsense Mutation: Inserts a stop codon, halting protein synthesis.

    • Missense Mutation: Changes one amino acid to another.

Point Mutation Illustrative Example: Sickle Cell Disease

• Cause: A missense mutation substitutes valine (non-polar) for glutamic acid (polar) in hemoglobin.

• Effect:

  • Causes hemoglobin molecules to stick together, leading to sickled red blood cells under low oxygen conditions.

  • Results in tissue damage and is a recessive condition.

  • Heterozygote Advantage: One copy of the mutation provides resistance to malaria.

Frameshift Mutations

• Caused by insertion or deletion of nucleotides, altering the reading frame of codons.

Germline Mutation

• Occur in cells that produce gametes, inherited by offspring and present in every cell of the organism.

• Can be passed on to future generations.

• Subject to natural selection (e.g., sickle cell anemia)

Somatic Mutations

• Occur in body cells, not passed to offspring.

• Example: Mutations from UV exposure causing skin cancer.

Vertical Gene Transfer

• Transmission of genetic material from parents to offspring, where organisms pass on all or half of their genome

• For example, a bacterium copying its entire genome during reproduction or humans passing genes through gametes.

Horizontal Gene Transfer

• When organisms get genes from other organisms (not their parents)

• In single-celled organisms these genes are passed on when they reproduce, but in multicellular organisms they must enter reproductive cells to be inherited.

Bacterial Conjugation

• Direct transfer of DNA (usually a plasmid) between two bacterial cells through a physical connection called a pilus, where the DNA is copied and passed to the recipient.

Bacterial Transformation

• Process in which a bacterium takes in free DNA from its environment and incorporates it into its own genome.

Viral Transduction

• Transfer of genetic material from one bacterium to another via a virus, when viral particles accidentally carry bacterial DNA and insert it into a new host cell.

Viral Recombination

• Process where two different viruses infect the same cell and exchange genetic material, producing new viral combinations.

Recombinant DNA

• DNA made by combining DNA from different species

• Made by restriction enzymes cutting DNA creating sticky ends and matching the ends base pair via hydrogen bonding. DNA ligase seals the bond to form a DNA molecule

Recombinant Plasmids

• Plasmid and foreign gene are cut with the same enzyme and if their sticky ends match ligase joins them and inserted into the bacteria

• Bacteria can now produce foreign proteins

Gel Electrophoresis

• Technique that separates DNA fragments by size using an electric current, where smaller fragments move farther through a gel.

• Longest are near negative charge

• Shortest are near positive charge

Restriction Site Mapping

• Process of identifying where restriction enzymes cut DNA by analyzing fragment sizes (often using gel electrophoresis).

PCR (Polymerase Chain Reaction)

• Method used to rapidly make millions of copies of a specific DNA sequence through repeated heating, cooling, and DNA replication.

DNA Sequencing

• Process of determining the exact order of nucleotides (A, T, C, G) in DNA.