Molecular Biology and Gene Expression Practice Flashcards

Introduction and Definitions of Molecular Biology

• Conceptual Overview: Molecular biology is the study of biology at a molecular level, specifically focusing on gene structure and function to understand the molecular basis of heredity, genetic variation, and gene expression patterns.
• Interdisciplinary Nature: The field overlaps significantly with other areas of biology and chemistry, notably genetics and biochemistry.
• Primary Focus Areas:
  • Understanding interactions between various cellular systems, including the relationships between DNA, RNA, and protein biosynthesis.
  • Learning how these molecular interactions are regulated.
  • The study of the formation, structure, and function of essential macromolecules (nucleic acids and proteins) and their roles in cell replication and the transmission of genetic information.
• The Three Molecules of Life: All life depends on three critical molecules: DNA, RNA, and proteins.
• The Life Alphabet: While the English alphabet has 26 letters, the "Life alphabet" contains only 4 letters: $A$, $T$, $G$, and $C$.
• Static vs. Dynamic Elements:
  • Structure: Considered static.
  • Function: Considered dynamic.

History of Genetic Discovery and Transmission Genetics

• Transmission Genetics: This field deals with the transmission of traits from parental organisms to offspring. The chemical composition of genes remained unknown until 1944.
• The Chromosome Theory of Inheritance: Chromosomes are the discrete physical entities carrying genes.
  • Thomas Hunt Morgan: Used the fruit fly, Drosophila melanogaster, to study genetics.
  • Autosomes: Occur in pairs in individuals.
  • Sex Chromosomes: Identified as $X$ and $Y$. Females possess two $X$ chromosomes, while males possess one $X$ and one $Y$.
• Genetic Loci and Phenotypes:
  • Locus: Every gene has a specific fixed place on a chromosome.
  • Genotype: The specific combination of alleles found in an organism.
  • Phenotype: The visible expression of the genotype.
  • Wild-type: The most common or standard phenotype.
  • Mutant Alleles: Usually recessive versions of the gene.
• 19th-Century Progress: The general structure of nucleic acids as long polymers of nucleotides linked by sugars through phosphate groups was discovered by the end of the 19th century.
• Key Historical Milestones:
  • Miescher (1869): Isolated nuclei from pus (white blood cells) and found a phosphorus-bearing substance called "nuclein," which is now known to be chromatin (DNA and chromosomal proteins).
  • Avery and colleagues (1944): Demonstrated that genes are composed of nucleic acids using a transformation test of S. pneumoniae (transition from avirulent to virulent strands). This work excluded protein and RNA as the chemical agents of transformation.
  • Hershey and Chase: Demonstrated that bacteriophage infection originates from DNA, not protein.

Relationship Between Genes and Proteins

• One Gene - One Enzyme Hypothesis: Defective genes produce defective or absent enzymes, leading to the early proposal that one gene is responsible for one enzyme.
• Corrections to the One Gene - One Enzyme Proposal:
  1. Enzymes may consist of several polypeptides; each gene codes for only one polypeptide.
  2. Many genes code for non-enzyme proteins.
  3. The end products of some genes are not polypeptides (e.g., rRNA, tRNA).
• Three Major Roles of Genes:
  1. Faithfully replicated.
  2. Direct the production of RNAs and proteins.
  3. Accumulate mutations to allow for evolution.

Chemical Composition and Structure of DNA

• The Watson-Crick Model (1953): DNA is a double helix with two complementary strands wound around each other.
• Components of a Nucleotide:
  1. Nitrogenous Base: Adenine ($A$), Guanine ($G$), Cytosine ($C$), and Thymine ($T$). In RNA, Uracil ($U$) replaces Thymine.
  2. Phosphoric Acid: Forms the phosphate group.
  3. Deoxyribose Sugar: Used in DNA. Carbon atoms in the sugar are noted as primed (e.g., $1'$, $5'$). Ribose sugar is used in RNA.
• Nucleoside vs. Nucleotide:
  • Nucleoside: Consists of a base and a sugar.
  • Nucleotide: Consists of a nucleoside plus at least one phosphate group attached via a phosphodiester bond.
• Purines and Pyrimidines:
  • Purines: Double-ring structures ($A$ and $G$).
  • Pyrimidines: Single-ring structures ($C$, $T$, and $U$).
• Phosphodiester Bonds: Phosphate groups join sugars in a chain through their $5'$ and $3'$ hydroxyl ($OH$) groups.
• Polarity:
  • 5' End: Free $5'$-phosphate group.
  • 3' End: Free $3'$-hydroxyl group.
• The Double Helix Geometry:
  • Backbone: Sugar-phosphate groups on the outside, linked by covalent bonds.
  • Interior: Bases aligned on the inside, connected by hydrogen bonds.
  • Directionality: Strands are antiparallel.
  • Measurements: Approximately 10 base pairs ($bp$) per turn. The helix has a uniform width because a purine always pairs with a pyrimidine.
• Base-Pairing Rules:
  • $A$ pairs with $T$ (DNA) or $U$ (RNA).
  • $G$ pairs with $C$.
  • Chargaff's Rules: In any organism, the ratio of $A$ to $T$ and $G$ to $C$ is fixed ($A=T$ and $C=G$).

Structural Variants of DNA

• B-DNA (Watson-Crick Form):
  • A right-handed helix (clockwise-turning).
  • Predominant form in vivo under high humidity ($95 \%$) and low salt.
  • Bases are perpendicular to the axis with $10.5$ bases per turn.
  • Wide major groove; narrow, moderate-deep minor groove.
• A-DNA:
  • A right-handed helix formed under low humidity ($75 \%$) and high salt.
  • Bases are tilted relative to the axis with $11$ bases per turn.
  • Deep/narrow major groove; shallow/broad minor groove.
  • RNA adopts an A-form helix in double-stranded regions because the 2'$-hydroxyl group on ribose hinders B-form formation.\n* **Z-DNA**:\n * A left-handed helix discovered by Alexander Rich in 1979.\n * Forms zig-zag backbones often in repeating GC sequences.\n * Contains 12 base pairs per turn.\n * Required for the activation of certain genes.\n\n# Physicochemical Properties of Nucleic Acids\n\n* **GC Content**: The total percentage of G+C$varies across organisms ($22 \%$to$73 \%$). Higher$GC content increases DNA density and thermal stability.\n* **DNA Melting and Denaturation**:\n * **Melting Temperature (T_m)**: The temperature at which DNA strands are half-denatured (dissociated).\n * **Denaturing Agents**: Heat, organic solvents, high pH, and low salt concentration.\n* **Renaturation (Annealing)**: The process of separated strands coming back together.\n * **Optimal Temperature**: Approximately 25^{\circ}\text{C}$below the$T_m.\n * **Other Factors**: Higher DNA concentration and increased time promote better annealing.\n* **Hybridization**: Putting together a combination of two different nucleic acids (e.g., DNA-RNA hybrid or two different DNA strands).\n* **DNA Size Expressions**:\n 1. Number of base pairs.\n 2. Molecular weight (average MW$for$1$base pair =$660 Daltons).\n 3. Length (33.2\,\text{Å}$per helical turn of$10.4\,bp).\n* **DNA Shapes**:\n * **Circular**: Typical of phage DNA.\n * **Linear**: Found in many organisms.\n * **Supercoiled**: Coils around itself like a twisted band.\n\n# DNA Extraction and Quantification\n\n* **Sources**: Biological evidence including blood, semen, saliva, urine, hair (root/shaft), teeth, bone, tissue, cigarette butts, stamps, and fingernails.\n* **Extraction Objectives**: Maximize DNA recovery and quality; remove inhibitors and nucleases.\n* **Common Extraction Procedures**:\n * **Organic (Phenol-Chloroform)**: Cell lysis $\rightarrow$ protein digestion with Sodium Dodecyl Sulfate (SDS) and Proteinase K $\rightarrow$ phenol-chloroform extraction $\rightarrow$ precipitation with ice-cold 95 \% ethanol and salt $\rightarrow$ wash with 70 \% ethanol.\n * **Non-Organic**: Salting out.\n * **Chelex**: Ion exchange resin.\n * **Silica-Based**: Qiagen-style exchange resins.\n* **Quantification by UV Absorbance**:\n * Aromatic bases absorb maximally at approximately 260\,nm.\n * **Standard Concentration**: 1.0\,A_{260} = 50\,\mu g/ml$for dsDNA and$38-40\,\mu g/ml for RNA/ssDNA.\n * **Beer-Lambert Law**: I = I_0 10^{-\epsilon dc}$where$I$is transmitted light,$I_0$is incident light,$\epsilon$is the molar extinction coefficient,$d$is path length, and$c is concentration.\n * **Optical Density (OD$)**:$OD_{\lambda} = \epsilon c.\n * **Purity Check**: An A_{260}/A_{280}$ratio of roughly$1.8 is considered good; lower values suggest protein contamination.\n* **Fluorometry**: Uses dyes like Hoechst 33258 that bind to the minor groove. Good for low concentrations (10-250\,ng/ml) and is not affected by rRNA or protein.\n\n# Visualizing DNA: Electrophoresis\n\n* **Principles**: Separation based on size as negatively charged DNA travels through a matrix (agarose or polyacrylamide) toward the cathode.\n* **Agarose Gels**:\n * Agarose is a polysaccharide polymer from seaweed.\n * Pore size is dictated by agarose concentration.\n * Standard gels separate fragments between 100\,bp$and$20,000\,bp.\n * **Pulsed-field gels**: Separate very large fragments (up to 10,000,000\,bp) by shifting the orientation of electric fields.\n* **Staining Methods**:\n * **Ethidium Bromide**: An anti-trypanosomal drug that intercalates into base pairs and fluoresces under UV light (302\,nm$or$260\,nm).\n * **SYBR Gold**: 10-fold more sensitive than ethidium bromide but expensive.\n * **Methylene Blue**: Non-toxic but less sensitive and time-consuming.\n * **Silver Staining**: High sensitivity but time-consuming and stains proteins.\n\n# DNA Replication\n\n* **General Features**: It is an anabolic polymerization process that is semiconservative (each new DNA molecule consists of one original parental strand and one daughter strand).\n* **Replication in Prokaryotes**:\n * **Origins**: Begins at a single origin of replication and proceeds bidirectionally.\n * **Enzymes**: \n * **Helicase**: Removes chromosomal proteins and unwinds the DNA.\n * **Gyrases/Topoisomerases**: Remove supercoils.\n * **DNA Polymerase III**: Main replicative enzyme; synthesizes DNA only in the 5'$to$3' direction.\n * **Primase**: Synthesizes an RNA primer.\n * **DNA Polymerase I**: Removes primers and fills gaps.\n * **DNA Ligase**: Joins fragments.\n * **Leading vs. Lagging Strands**: The leading strand is synthesized continuously; the lagging strand is synthesized discontinuously in Okazaki fragments.\n* **Replication in Eukaryotes**:\n * Uses four different DNA polymerases.\n * Thousands of replication origins per chromosome.\n * Shorter Okazaki fragments.\n * Plant/animal cells methylate only cytosine bases.\n\n# Polymerase Chain Reaction (PCR)\n\n* **Definition**: A technique used to amplify specific segments of DNA into millions of copies.\n* **Components**: DNA template, primers, nucleotides (dNTPs), heat-stable DNA polymerase (e.g., Taq), buffer solution, and a thermal cycler.\n* **The Three Main Steps**:\n 1. **Denaturation**: Heating to 94-98^{\circ}\text{C}$for$30-40 seconds to separate strands.\n 2. **Annealing**: Lowering temperature to 50-65^{\circ}\text{C}$for$20-40 seconds to allow primers to bind.\n 3. **Extension**: Raising temperature to 72^{\circ}\text{C}$for$1-2 minutes; DNA polymerase adds nucleotides to the primers.\n* **Applications**: Medical diagnostics, forensics, and genetic testing.\n\n# Gene Expression: Transcription\n\n* **Process Overview**: Using genetic instructions in DNA to synthesize RNA. Transcription is enzyme-mediated by RNA polymerase.\n* **Phases of Transcription**:\n 1. **Initiation**: RNA polymerase binds to a promoter (upstream of the gene). It causes localized melting of the DNA and forms the first few phosphodiester bonds.\n 2. **Elongation**: RNA polymerase moves along the template, linking ribonucleotides in the 5'$to$3' direction. A transcription "bubble" of separated DNA moves with the enzyme.\n 3. **Termination**: Specific regions (terminators) cause the RNA product and polymerase to dissociate from the DNA.\n* **Directionality**: RNA sequences are written 5'$to$3'. The promoter area is "upstream," and the gene is "downstream."\n* **Differences from Replication**:\n * Transcription makes only one RNA strand (asymmetrical), while replication is semiconservative.\n * DNA melting is transient in transcription but permanent in replication.\n* **RNA Processing (Eukaryotes)**: Extensive processing in the nucleus before translation, including capping (5'$end), poly-A tailing ($3' end), and splicing (removing introns, joining exons).\n\n# Gene Expression: Translation\n\n* **Ribosomes**: The protein-synthesizing machines comprised of RNA and protein. \n * **Bacteria (70S)**: Consists of a 50S$large subunit and a$30S small subunit. \n * "S" stands for the sedimentation coefficient measured in an ultracentrifuge.\n* **Transfer RNA (tRNA)**: The adapter molecule proposed by Crick. \n * **Structure**: Cloverleaf model.\n * **Function**: One end binds a specific amino acid; the other end contains a 3-bp **anticodon** that pairs with a **codon** in mRNA.\n * **Enzyme**: Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to tRNA.\n* **Phases of Translation**:\n 1. **Initiation**: The start codon (AUG) interacts with methionyl-tRNA (eukaryotes) or N-formylmethionyl-tRNA (bacteria). The Shine-Dalgarno sequence (bacteria) attracts ribosomes.\n 2. **Elongation**: Requires Elongation Factor EF-Tu$and energy from$GTP. The first tRNA occupies the **P site**; the incoming tRNA binds to the **A site**. **Peptidyl transferase** forms the bond.\n 3. **Termination**: Release factors recognize stop codons (**UAG**, **UAA**, **UGA**) and release the polypeptide.\n* **mRNA Structure**: Contains a 5'$leader (5'-UTR), an open reading frame ($ORF$), and a trailer ($3'-UTR).\n\n# Regulation of Gene Expression and Mutations\n\n* **Operons (Prokaryotes)**: Systems including a promoter, an operator (on/off switch), and structural genes. The **lac operon** (breaks down lactose) was one of the first discovered.\n* **Regulation Levels**: Expression can be controlled by chromatin modifications, transcription factors, enhancers, RNA stability, or utilization.\n* **Mutations**: Changes in nucleotide sequences essential for evolution.\n * **Silent Mutation**: Changes the codon but results in the same amino acid.\n * **Conservative Mutation**: Results in a chemically similar amino acid.\n * **Non-conservative Example**: In sickle cell disease, a glutamate codon (GAG$) changes to a valine codon ($GUG$$), causing significant structural changes in the protein product.