Fundamental Cellular Processes: DNA, Gene Expression, Energy

Posted on Jun 22, 2025 in Biology

DNA Replication In Vitro: Polymerase Chain Reaction

DNA Replication: Parental strands separate, complementary strands are made, and daughter strands are created.

Polymerase Chain Reaction (PCR): A method of making copies, allowing a targeted region of DNA to be replicated (amplified) into as many copies as desired. PCR requires:

• Template DNA
• DNA Polymerase
• Four bases (dNTPs)
• Two primers

This process makes 2ⁿ copies.

Oligonucleotides: Primer sequences, typically 20-30 nucleotides long, are added so the number of primer DNA molecules is greater than the number of template DNA molecules. They flank the region of DNA to be amplified.

PCR Steps:

1. Denaturation: Melting the solution in the tube to break hydrogen bonds, separating the DNA strands.
2. Annealing: The solution is cooled, allowing the two primers to bind to their complementary DNA segments.
3. Extension: The solution is heated to the optimal temperature for DNA Polymerase. The polymerase elongates each primer, synthesizing new DNA strands.

DNA Replication In Vivo: Cellular Mechanisms

DNA Polymerase reads the sequence on a template strand in the 3’ to 5’ direction and synthesizes new DNA in the 5’ to 3’ direction. It cannot start on its own; it must start from an existing 3’OH end (a primer made from RNA).

Polymerization: Requires an enzyme and a form of energy (commonly triphosphates). DNA replication is semiconservative: resulting in half parent and half daughter strands. Human cells take longer to replicate, so they have multiple origins of replication. In bacteria, DNA is circular.

Key Enzymes and Proteins in DNA Replication:

• Helicase: Breaks hydrogen bonds, unwinding the DNA double helix.
• Primase: Synthesizes RNA primers on both leading and lagging strands.
• DNA Polymerase I: Replaces RNA primers with DNA nucleotides.
• DNA Ligase: Catalyzes phosphodiester bond formation, joining DNA fragments; joins leading and lagging strands at the Origin of Replication (OriR).
• Topoisomerase: Relaxes supercoiled DNA.
• SSBPs (Single-Strand Binding Proteins): Coats single-stranded DNA to prevent re-annealing.
• DNA Polymerase III: Synthesizes DNA in the 5’ to 3’ direction on both leading and lagging strands.

Replication Bubble: Consists of two replication forks. There is one origin of replication for both strands in prokaryotes, while eukaryotes have multiple OriRs.

Three Problems in DNA Replication and Their Solutions:

1. Problem: Separating DNA strands only a little at a time. Solution: Helicase unwinds DNA at the replication fork.
2. Problem: Making primers for DNA Polymerase. Solution: RNA Polymerase / RNA Primase makes RNA primers.
3. Problem: Allowing synthesis to happen simultaneously from the two template strands. Solution: Synthesis occurs in opposite directions, but overall must progress on both strands in the direction of the growing replication fork.

dNTPs: Deoxyribonucleotide triphosphates (bases: A, T, G, C).

Okazaki Fragments: Fragments of the lagging strand, resulting from discontinuous replication.

Leading Strands: Replicated continuously.

Translation & Electron Micrographs: Bacterial vs. Eukaryotic

Translation in bacteria versus eukaryotes differs in location due to the presence of a nucleus in eukaryotes.

Electron Micrographs: Longer ribosomes indicate earlier binding. They capture protein synthesis in the cytoplasm.

• Bacteria: Will show DNA and RNA being transcribed and translated at the same time (coupled transcription-translation).
• Eukaryotes: Will only show DNA being transcribed or mRNA being translated, as these processes are spatially separated.

Transcription: Occurs in the 3’ to 5’ direction of the template strand (new nucleotides are added to the 3’ end of the growing RNA molecule, with the 3’ end inside the RNA Polymerase).

Translation: Occurs in the 5’ to 3’ direction of the mRNA (new amino acids are added to the carboxyl end of the growing polypeptide chain).

DNA Mutations: Types, Causes, and Consequences

Most mutations are spontaneous (without an external cause).

• Wild Type: Found in nature.
• Genotype: Genetic information.
• Phenotype: Expressed qualities.

Substitution: Mutation rates differ among organisms. Hotspots are especially mutable areas. The rate of mutation depends on germ cells (where DNA repair is more efficient) and somatic cells. For a mutation to be inherited, it must escape the repair system.

• Point Mutation: A base pair is replaced with another; this is the most frequent type and can be silent (no change in amino acid).
• Missense Mutation: A point mutation that causes a new amino acid to be incorporated.
• Nonsense Mutation: A point mutation that causes a premature stop codon.
• Single-Nucleotide Polymorphism (SNP): A site in the genome where a base pair differs among individuals in a population.

Insertions and Deletions:

• Non-coding: Have little effect.
• Coding: The effect depends on size, but when not in multiples of three, they have huge consequences, leading to frameshift mutations.

Gene Expression Regulation: Operon Systems

A gene is expressed when a gene product is being actively synthesized and used.

Constitutively Expressed Genes: Expressed all the time, though levels can vary. Control mechanisms include:

• Promoter Strength: How efficiently RNA Polymerase and transcription factors bind to the promoter determines the frequency of transcription and how many mRNA molecules are made.
• mRNA Half-Life: How quickly mRNA is degraded after being made.

Environmentally Regulated Genes: Expressed only under certain conditions. Critical for efficient use of energy and survival. Regulation involves:

1. DNA Regulatory Region (Operator): Located near the promoter.
2. Regulatory Proteins: Genes specifying regulatory proteins are not part of the operon itself. They are usually constitutively expressed at low levels.
3. Signal Molecules: (e.g., MalT, LacI) Affect the binding of regulatory proteins (allosterically) to regulatory regions. These can be inducers (for CATABOLIC pathways) or co-repressors (for ANABOLIC pathways).

• Catabolic: Break down molecules.
• Anabolic: Build up molecules.

Operons: Regions of bacterial DNA that code for functionally related genes. They are transcribed from a single promoter to a polycistronic mRNA (one mRNA but multiple proteins due to multiple start/stop codons). Operons often contain environmentally regulated genes.

Types of Regulation:

• Positive Regulation: (Often with a weak promoter) A regulatory gene product (protein) activates gene expression by binding to a DNA regulatory region. This protein is called an activator. (Note: The regulatory region is not part of the operon itself).
• Negative Regulation: (Often with a strong promoter) A protein inhibits gene expression through a repressor.

Gene Regulation in Prokaryotes: Simpler than in eukaryotes because DNA is not in nucleosomes, mRNA is not processed, and there is no separation of transcription and translation (both are subject to regulation).

Polycistronic mRNA: When coding sequences are transcribed from a promoter into a single sequence of mRNA.

Nomenclature: Genes are typically italicized and lowercase (e.g., malT, lacZ, lacA). Proteins are typically capitalized (e.g., LacZ, LacY, MalT).

Mal Operon: Positive Regulation

Maltose (a disaccharide) is a source of energy and carbon for growing cells. Enzymes required to break it down are clustered in an operon.

Features:

• Regulatory Region/Promoter Region: RNA Polymerase binds here.
• Operator Region: Upstream and close to the malPQ genes. MalT, a positive regulator (activator protein), binds here.
• Regulatory Protein: MalT.
• Signal Molecule: Maltose.

Transcription expression is under the control of two DNA-binding proteins: RNA Polymerase and MalT. Increased maltose leads to increased malPQ expression. Without maltose, MalT has poor binding affinity to the operator. When MalT is more stably bonded (in the presence of maltose), it helps RNA Polymerase bind to the operator.

Lac Operon: Negative Regulation

Lactose consists of glucose and galactose. The lac operon contains lacZ, lacY, and lacA genes (which create related proteins). Lac operon expression increases when lactose increases. Lactose binds to the repressor protein. The promoter for the lac operon is strong and binds RNA Polymerase strongly. For the lac operon, the operator is downstream and overlaps the promoter. LacI (repressor protein) can bind to the promoter (it is constitutively expressed).

• lacZ: Cleaves the lactose molecule into glucose and galactose components. lacZ+ indicates a functional, non-mutant lacZ gene.
• lacY: Transports lactose into the cell, with more being made when lactose is present. lacY+ indicates a functional gene.

These are structural genes. Mutants that can always transcribe (even without lactose) are called constitutive mutants. Lactose acts as an inducer.

• Low Basal Levels: Occur in the absence of an inducer.
• High Levels: Occur in the presence of an inducer.
• Zero Expression: Due to a significant mutation (e.g., deletion) or absence of DNA regulatory proteins that can make the operon inactive.

Catabolic Operon: Produces proteins involved in the catabolism of a signal molecule.

Anabolic Operon: Proteins produced by the operon are used to synthesize a signal amino acid. If the signal molecule is present, the cell does not need to synthesize it. If the signal molecule is absent, high levels of transcription occur.

An example of an anabolic operon is the arginine operon.

Photosynthesis is anabolic (reduction, uses energy). Cellular respiration is catabolic (oxidation, produces energy).

Cellular Respiration & Redox Reactions

Cells use ATP (adenosine triphosphate). Carbon-based compounds are often a stable form of energy storage.

• Phototrophs: Harvest energy from the sun.
• Autotroph: Self-feeder (regarding carbon).
• Chemotrophs: Obtain energy from chemical compounds.
• Heterotroph: Other-feeders (regarding carbon).

Metabolism: The set of chemical reactions that convert molecules and transfer energy.

• Catabolism: Breaks down molecules, produces ATP.
• Anabolism: Builds up molecules, uses ATP.

Chemical Energy: Energy potential (Ep) stored in bonds. Decreased energy potential leads to increased stability.

Redox Reactions: Shifting energy potential in chemical bonds.

• LEO (Loss of Electrons is Oxidation): Gains Oxygen / Loses Hydrogen, Releases Energy, Breaks Down Molecules.
• GER (Gain of Electrons is Reduction): Gains Hydrogen / Loses Oxygen, Takes Energy, Builds Up Molecules.

High energy potential is stored in reduced carbon. More oxidized molecules are more stable.

Adenosine: The base of ATP, consisting of Adenine + a 5-carbon sugar ribose + triphosphate. Energy is held in the bonds between phosphate groups.

Gibbs Free Energy (G): Energy available to do work.

• G > 0: Products have more energy, energy is required, endergonic reaction.
• G < 0: Energy is released, exergonic reaction.

H (Total Energy) = G + TS (Energy lost to entropy).

Energetic Coupling: A spontaneous reaction drives a non-spontaneous reaction. The net G < 0, and the reactions must occur together.

Cellular Respiration: Key Stages & Processes

Cellular Respiration: Glucose is oxidized to CO2, converting stored energy to ATP (a catabolic process).

1. Glycolysis (Cytoplasm): Glucose is partially broken down to produce pyruvate; energy is transferred to ATP and to reduced electron carriers (NADH).
2. Pyruvate Oxidation (Cytoplasm/Mitochondrial Matrix): Pyruvate is oxidized to acetyl coenzyme A (acetyl-CoA), producing reduced electron carriers (NADH) and releasing CO2.
3. Citric Acid Cycle (TCA) (Mitochondrial Matrix): The acetyl group is completely oxidized, producing ATP, NADH, and FADH2, and releasing CO2.
4. Oxidative Phosphorylation (Cell Membrane/Mitochondrial Membrane): Reduced electron carriers from stages 1-3 donate electrons to the electron transport chain, and much ATP is produced.

Nutrients: Electron donors (lose electrons, become oxidized).

Electron Acceptors: Gain electrons, become reduced, and capture energy loss from electron donors.

• Aerobic Respiration: Oxygen is consumed as the final electron acceptor.
• Anaerobic Respiration: Occurs in the absence of oxygen.

Substrate-Level Phosphorylation: A phosphate group is transferred directly to ADP from an organic molecule (catalyzed by an enzyme in steps 1 and 3 of glycolysis/fermentation).

Oxidative Phosphorylation: Energy is first transferred to electron carriers (which carry electrons and energy from one reaction to another). These carriers transport energy to the electron transport chain (which transfers electrons along membrane proteins to a final electron acceptor), which then harnesses the energy to produce ATP (stage 4).

In anaerobic respiration, glucose is oxidized, releasing CO2, and an inorganic molecule (not oxygen) is reduced.

Glycolysis & Fermentation Pathways

Glycolysis: (Forms: 2 ATP, 2 NADH, 2 Pyruvate) Partial oxidation of glucose. This is a catabolic process.

1. Investment/Cleavage Phase: Two phosphate groups are added to glucose. This requires energy (2 molecules of ATP) and is an endergonic process. This phosphorylation traps the phosphorylated glucose in the cell and destabilizes it for the next steps due to negative charge repulsion.
2. Cleavage: The 6-carbon molecule is cleaved into two 3-carbon molecules (G3P).
3. Energy Payoff Phase: 4 molecules of ATP (via substrate-level phosphorylation) and 4 electrons are removed from G3P to form 2 molecules of NADH (which contributes to ATP synthesis during oxidative phosphorylation). The process ends with 2 molecules of pyruvate.

Fermentation: (Forms: Lactic Acid or Ethanol) Extracts energy from glucose without oxygen. Uses an organic molecule as an electron acceptor. Involves the breakdown of pyruvate. Converts sugars to acids, gases, or alcohols. Bacteria, yeast, and muscle cells can ferment. All ATP is made by substrate-level phosphorylation (pyruvate cannot enter mitochondria as it lacks a terminal electron acceptor, such as O2). There is no net oxidation of carbon; electrons removed from some carbon atoms are returned to others. Products of fermentation are waste products. To ferment, NADH is oxidized back to NAD+, which then returns to glycolysis. Electrons are donated to create waste products, using organic molecules.

• Lactic Acid Fermentation: Occurs in animals and bacteria. Electrons from NADH are transferred to pyruvate to produce lactic acid and NAD+.
• Ethanol Fermentation: Occurs in plants and fungi. Pyruvate releases CO2, forming acetaldehyde. Electrons from NADH are then transferred to acetaldehyde to make ethanol and NAD+. (This process also produces 2 CO2 per glucose).

Respiration: ATP is made in stages 1, 3, and 4. It uses O2 to respire and makes most ATP by oxidative phosphorylation.

Acetyl-CoA Synthesis & Citric Acid Cycle

Pyruvate Processing: (Forms: 1 NADH, 1 CO2, 1 Acetyl-CoA per pyruvate) Even after glycolysis (anaerobic), pyruvate contains energy potential. With oxygen, pyruvate can be further oxidized, first to acetyl-CoA, then through further reactions in the citric acid cycle. Pyruvate oxidation links glycolysis to the citric acid cycle.

Process: Pyruvate is oxidized and splits to form CO2. Two electrons are lost and donated to NAD+, forming NADH. Coenzyme A is added to the molecule, forming Acetyl-CoA.

Citric Acid Cycle (Krebs Cycle / TCA Cycle): (Forms per glucose, after 2 turns: 2 ATP, 6 NADH, 2 FADH2, 4 CO2) All carbons are completely oxidized (acetyl group to CO2), and energy is transferred to ATP by substrate-level phosphorylation, plus electron carriers NADH and FADH2. Thus, the citric acid cycle provides electrons to the electron transport chain. It takes place in the mitochondrial matrix and is composed of 8 reactions. The last reaction regenerates oxaloacetate.

Acetyl-CoA is an output of pyruvate processing and an input for the citric acid cycle.

Electron Transport Chain & Oxidative Phosphorylation

How is the energy in NADH and FADH2 used to synthesize ATP? It is released in a series of redox reactions that occur as electrons pass through the electron transport chain.

Oxidative Phosphorylation: (Forms: ~2.5 ATP per NADH, ~1.5 ATP per FADH2; Total ~28 ATP, H2O) Involves membrane-bound enzymes and a proton (H+) gradient that drives ATP phosphorylation. In eukaryotes, it occurs in the mitochondrial inner membrane (the H+ gradient is in the intermembrane space). In bacteria, it occurs on the plasma membrane.

1. Electrons are derived from high-energy intermediates NADH and FADH2. NADH donates 2 electrons to Complex I, and FADH2 donates 2 to Complex II.
2. Energy is captured as electrons pass along the electron transport chain in a step-down process. Electrons ultimately end up on oxygen (the Terminal Electron Acceptor, TEA), and O2 is reduced to H2O.
3. This process drives the energy needed to pump protons across the mitochondrial membrane. H+ moves from the matrix to the intermembrane space.
4. Protons flow through ATP synthase, resulting in indirect synthesis of ATP (in the matrix). Protons are pumped to the intermembrane space from the matrix. ATP synthase (its stem rotates as H+ diffuse through, providing energy for the head to phosphorylate ADP) converts proton gradient energy to ATP.

In bacteria, protons are pumped to the outside of the cell membrane.

Coenzyme Q and Cytochrome C are electron carriers involved in the ETC.

This all results in proton pumping (accumulation of protons (H+) in the intermembrane space), creating an electrochemical proton gradient.

Anaerobic Respiration: Uses an inorganic terminal electron acceptor on the electron transport chain (e.g., Nitrate or Sulfate). Inorganic compounds are used for the electron source instead of glucose.

Photophosphorylation & The Calvin Cycle

Photosynthesis: Anabolic process where carbohydrates are synthesized from CO2 and H2O. Requires energy from the sun. It is a redox reaction; the ultimate electron donor is H2O. It involves the conversion of light energy to chemical energy and the reduction of carbon (which requires energy).

1. Light-Dependent Reactions (Photophosphorylation):
2. Light-Independent Reactions (Calvin Cycle):

Photophosphorylation (Light-Dependent): (Forms: ATP, NADPH, O2) This is the photosynthetic electron transport chain. Water is oxidized to produce O2 (donates electrons to the ETC). NADP+ is reduced to form NADPH. ATP is synthesized via a proton gradient driven by chemiosmosis (proton concentration in thylakoids).

In chloroplasts, the first light-dependent reactions occur in the thylakoid membrane. Chloroplasts make lots of energy (ATP) and high-energy intermediates (NADPH) via an ETC called photophosphorylation. These are made for the next set of reactions in the Calvin Cycle. Cells use chloroplasts to convert light energy into high-energy intermediates, accomplished by photophosphorylation, an ETC which has two photosystems, often referred to as a Z-scheme. Bacteria do not have chloroplasts but have photosynthetic internal membranes.

Z-Scheme

(Input: Photons and H2O; Output: ATP, NADPH, O2) Photosystems I and II work together in the thylakoid membrane.

1. Electrons are supplied by H2O.
2. Light energy is required for PSII to strip electrons from water (splitting the water).
3. Electrons move along the ETC to provide energy to pump protons (H+) into the thylakoid lumen.
4. An electrochemical gradient is created in the thylakoid lumen.
5. More light energy input at PSI energizes electrons to drive the reaction of NADP+ to NADPH.
6. NADPH is synthesized in the stroma and will be used in the Calvin Cycle.
7. ATP is synthesized in the stroma via chemiosmosis as H+ moves through ATP synthase.

This is called oxygenic photophosphorylation (oxygen is a product). Photophosphorylation does not have a terminal electron acceptor (TEA) and electron donors (like glucose). Electrons end up on a molecule already present (e.g., NADP+, which is not a TEA). “Terminal” refers to a molecule that has been taken up by the cell from the environment.

Calvin Cycle (Light-Independent)

The Calvin Cycle uses these ATP and NADPH to synthesize organic carbon from inorganic CO2. This process is also called carbon fixation. These carbons can now leave the chloroplast and be used in cellular respiration or to build other macromolecules.

NADPH: A high-energy intermediate synthesized in photophosphorylation and then used in anabolic reactions. Similar to NADH but with an extra phosphate group. Having NADH and NADPH in cells allows for cells to differentiate between two electron carriers and their roles.

The chloroplast is within the cytosol of a cell. The cytosol is the location of the Calvin Cycle. The chloroplast is the location of the light-dependent reactions. In a plant cell, chloroplast and mitochondria are linked processes. Carbohydrates (reduced form of carbon) are produced by chloroplasts from CO2. Sugars undergo catabolism (cellular respiration) to produce energy in mitochondria.

• Redox for Photosynthesis: Reducing carbon.
• Cellular Respiration: Oxidizing carbon.

Nitrate and Sulfate are common electron acceptors in species that live in oxygen-poor environments. Inorganic compounds such as H2 and H2S are used as electron sources instead of glucose.

Thylakoid Membrane: The folded center of the chloroplast. The region surrounding it is the stroma. When chlorophyll absorbs light, it excites electrons. The Cytochrome complex is another protein complex in the chain, in addition to photosystems.

How protons build up in the thylakoid membrane: Oxidation releases O2 + protons (H+); the cytochrome complex + plastoquinone act as proton pumps. Protons build up in the lumen.

ATP Hydrolysis: Breaking off one phosphate from ATP. This reaction is coupled with endergonic reactions to make them possible. A weak and unstable bond is broken, leading to an increase in stability.