BIO 215: DNA and Gene Expression

Biological Perspective and Fundamental Definitions

• Role of DNA in Cellular Success:
  • DNA (Deoxyribonucleic acid) serves as the primary repository of genetic instructions required for the synthesis of proteins.
  • Proteins fulfill critical roles as both structural components of the cell and as enzymes.
  • Enzymes are responsible for mediating essential cellular activities, including biosynthesis and energy production.
  • DNA carries the comprehensive information set necessary for the creation and functional operation of a cell.
• Key Genetic Terminology:
  • Gene: A specific region of DNA that contains the code for a single protein.
  • Genome: The complete set of genetic information within an organism, encompassing both the chromosome and any plasmids.
  • Genomic Composition: While the genomes of all cellular life are composed of DNA, certain viruses utilize an RNA genome.

Detailed Structure of DNA and Nucleotides

• Nucleotide Composition: DNA is a polymer composed of subunits called nucleotides. Each nucleotide consists of three functional groups:
  • Pentose Sugar: Specifically deoxyribose in DNA.
  • Phosphate Group: Attached to the sugar molecule.
  • Nitrogenous Base: Four distinct bases are used: Adenine (A), Thymine (T), Guanine (G), and Cytosine (C).
• Chemical Bonding and Polarity:
  • Phosphodiester Bonds: Nucleotides are linked together covalently to form a strand.
  • Strand Directionality: A single strand of nucleotides possesses distinct ends, beginning at the $5'$ end and terminating at the $3'$ end.
  • Sugar-Phosphate Backbone: The repeating sugar and phosphate units form the structural backbone of the DNA strand, with nitrogenous bases protruding from it.
• The Double Helix Structure:
  • Hydrogen Bonding: Nucleotide strands are joined to one another via hydrogen bonds shared between complementary nitrogenous bases.
  • Complementary Base Pairing: Specificity of binding ensures that Adenine (A) always pairs with Thymine (T), and Guanine (G) always pairs with Cytosine (C).
  • Complementary Nature: For every base present on one strand, there is a corresponding partner base on the second strand capable of bonding.
  • Antiparallel Arrangement: The two strands are oriented in opposite directions; the $5'$ end of one strand aligns with the $3'$ end of the opposing strand.
  • Helical Geometry: The interaction between these strands creates a twisted, helical structure.

RNA Structure and Comparison to DNA

• Sugar Component: RNA utilizes ribose instead of the deoxyribose used in DNA.
  • Ribose differs from deoxyribose by the presence of an additional hydroxyl ($OH$) group.
• Nitrogenous Bases: RNA utilizes Uracil (U) in place of Thymine (T). Uracil pairs with Adenine.
• Physical Properties:
  • RNA is typically single-stranded, whereas DNA is double-stranded.
  • RNA molecules are generally much shorter in length compared to the substantial length of chromosomal DNA.

DNA Replication: Process and Mechanics

• Biological Necessity: DNA replication is essential for cell multiplication, as every daughter cell requires its own complete chromosome.
• Semiconservative Replication:
  • This mechanism ensures that after replication, each new chromosome consists of one original (parental) strand and one newly synthesized (daughter) strand.
  • Each original strand serves as a template for the construction of the new complementary strand.
• Initiation and Progression:
  • Origin of Replication: Synthesis of chromosomal DNA begins at a specific site called the origin of replication.
  • Bidirectional Replication: Synthesis proceeds in both directions from the origin.
  • Replication Forks: This bidirectional movement creates two advancing forks where active DNA synthesis occurs.
  • Termination: The replication forks eventually meet at a designated terminating site to complete the process.
• Enzymatic Activity:
  • DNA Polymerases: These enzymes are responsible for synthesizing DNA.
  • Nucleotide Addition: Daughter strands are synthesized one nucleotide at a time.
  • Direction of Synthesis: Nucleotides can only be added to the $3'$ end of the growing molecule. Consequently, DNA synthesis always occurs in a $5' \rightarrow 3'$ direction.

Gene Expression and the Central Dogma

• Definition of Gene Expression: The process where a gene is utilized as a template to facilitate the synthesis of a protein.
• The Central Dogma of Molecular Biology: Represents the flow of genetic information: $DNA \rightarrow RNA \rightarrow Proteins$.
• Transcription: The process of synthesizing RNA from a DNA template.
• Translation: The process where the information encoded on a messenger RNA (mRNA) transcript is deciphered to synthesize a specific protein.

Transcription: Synthesis of mRNA

• Role of RNA Polymerase: This enzyme synthesizes a strand of RNA that is both complementary and antiparallel to the target gene on the DNA.
• Messenger RNA (mRNA): The resulting RNA strand is termed mRNA, which carries the genetic "photocopy" of instructions from the DNA (located in the nucleus or nucleoid) to the ribosomes (located in the cytoplasm) for protein production.
• The Process of Transcription:
  1. Binding: RNA polymerase binds to a DNA sequence known as the promoter, which determines which gene is to be expressed.
  2. Strand Separation: The DNA double strands are separated into a minus (-) strand (template) and a plus (+) strand.
  3. Elongation: RNA polymerase moves along the minus (-) strand, creating a complementary RNA piece in the $5' \rightarrow 3'$ direction.
  4. Termination: When the enzyme reaches a sequence called the terminator, RNA polymerase detaches from the DNA, and the mRNA transcript is released.

The Genetic Code and Translation

• The Codon: A series of three nucleotides on the mRNA that correlates to one specific amino acid in the protein chain.
• Translational Components: Requires mRNA, ribosomes, and transfer RNA (tRNA).
• Degeneracy and Redundancy:
  • There are 4 possible nitrogenous bases (A, C, G, U) arranged in 3-base triplets, resulting in $4 \times 4 \times 4 = 64$ possible codons.
  • There are only 20 amino acids used in proteins. Therefore, many amino acids are coded for by more than one codon.
  • Degenerate Code: This term describes the redundant nature of the code. For example, Leucine is coded by six different codons: UUA, UUG, CUU, CUC, CUA, and CUG.
• Universality: The genetic code is universal across almost all organisms. This allows a bacterium to produce a human protein (like insulin) if the human gene is inserted into its genome.
• Steps of Translation:
  1. Initiation: The ribosome binds to the $5'$ end of the mRNA. Upon reaching the start codon (AUG), an initiation complex forms consisting of the ribosome, a tRNA carrying methionine, and initiation factors.
  2. Elongation: A tRNA with an anticodon complementary to the mRNA codon arrives, carrying the next amino acid. Peptide bonds form between amino acids as the ribosome moves down the mRNA.
  3. Termination: The process ends when the ribosome reaches a stop codon (UAA, UAG, or UGA), which is not recognized by tRNA. The ribosome then dissociates from the mRNA.
• Transfer RNA (tRNA) Specifics: tRNA possesses an anticodon (e.g., CGG) that binds to a complementary codon (e.g., GCC) on the mRNA to deliver the correct amino acid (e.g., Proline/Pro).

Regulation of Bacterial Gene Expression

• Energy Conservation: Cells only synthesize proteins when needed to avoid wasting energy. For instance, enzymes for lactose degradation should only be produced if lactose is present in the environment.
• Types of Enzymes Based on Regulation:
  • Constitutive Enzymes: Synthesized constantly; these are usually critical for central metabolic pathways.
  • Inducible Enzymes: Synthesized only when their specific substrate is present (e.g., the lac operon).
  • Repressible Enzymes: Synthesis is halted when the final product of the pathway is already present in sufficient quantities.

The lac Operon Model

• Structure: A section of DNA comprising a promoter, an operator, and three structural genes: lacZ ($eta$-galactosidase), lacY (permease), and lacA (transacetylase).
• Mechanism in Absence of Lactose:
  • A repressor protein binds to the operator site.
  • This physical barrier blocks RNA polymerase from transcribing the genes; thus, no enzymes are made.
• Mechanism in Presence of Lactose:
  • Some lactose is converted into allolactose.
  • Allolactose acts as an inducer, binding to the repressor and changing its shape so it cannot bind to the operator.
  • RNA polymerase can then proceed with transcription, and the enzymes are produced (provided glucose is not available).

Signal Transduction and Environmental Response

• Signal Transduction: The ability of bacterial cells to sense environmental changes and adjust metabolism accordingly.
• Two-Component Regulatory System:
  1. Sensor Protein: Spans the cytoplasmic membrane and becomes phosphorylated ($P$) in response to an external stimulus.
  2. Response Regulator: An internal protein that receives the phosphate group from the sensor and becomes activated to either activate or repress specific genes.
• Quorum Sensing:
  • Organisms detect the density of their own population.
  • Gene expression is activated only when a "critical mass" of cells is reached.
  • Example: Pseudomonas aeruginosa only produces biofilms (contributing to disease) once a critical mass is achieved.

Natural Selection and Variation

• Antigenic Variation: The random alteration of surface proteins (found on flagella, pili, or membranes) within a population.
  • Example: Neisseria gonorrhoeae alters its pilin proteins. This allows some members of the population to evade the host's immune system as established antibodies will not recognize the new surface proteins.
• Phase Variation: The routine switching on and off of certain genes within a population. This creates metabolic variety, increasing the likelihood that some members of the population will survive changing environmental pressures.