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Polymerase & Bases
| Question | Answer |
|---|---|
| Central Dogma | describes the directional flow of genetic information: DNA is transcribed into RNA, and RNA is translated into protein. This process ensures that hereditary information stored in DNA is expressed into functional molecules that drive cellular activity. |
| High Fidelity of DNA Replication | DNA replication is highly accurate because of strict base-pairing rules, proofreading by DNA polymerases, and mismatch repair systems. This fidelity is essential to prevent harmful mutations and maintain genome stability across generations. |
| DNA Polymerases | enzymes that synthesize DNA strands in the 5′→3′ direction using existing strand as template and require primer for repli start. In bacter, Pol III is primary replicative enzyme; in humans, polymerases (α, δ, ε) do leading/lagging-strand synthesis. |
| RNA Polymerase | RNA polymerases transcribe RNA from DNA templates, but unlike DNA polymerases, they lack proofreading activity and are more error-prone. Bacteria have a single RNA polymerase, whereas humans use three distinct types. |
| Nucleotides | a compound consisting of a nucleoside linked to a phosphate group. Nucleotides form the basic structural unit of nucleic acids such as DNA. Consist of a pentose sugar + a nitrogenous base + and a phosphate. |
| Why is DNA stable and RNA unstable? | DNA has a deoxyribose sugar and RNA has a ribose sugar, which is less stable due to the extra hydroxyl group. |
| Polynucleotides | Long chains of nucleotides, the building blocks of DNA and RNA. The nucleic acids DNA and RNA are polynucleotides. |
| Nucleosides | Nucleotides without phosphate (nucleotides = nucleosides + phosphate). |
| Prime notation | Refers to carbon positions on the ribose/deoxyribose ring |
| Backbone of DNA and RNA | Connected by phosphodiester bonds between 5' and 3' hydroxyl groups. This linkage provides structural stability and directionality to the nucleic acid strand, giving it a 5′→3′ polarity. |
| Structures of DNA and RNA | know what they both look like and how to, tentatively, draw them. |
| Nitrogenous Bases | organic molecules with nitrogen atoms that are fundamental components of nucleic acids like DNA and RNA. |
| DNA bases | Adenine (A), Cytosine (C), Guanine (G), Thymine (T) |
| RNA bases | Uracil (U) replaces thymine (differs by one methyl group) |
| Hydrogen Bonds in Each Base pairing | Hydrogen bonds between complementary bases. C≡G: 3 hydrogen bonds (strong). A=T/U: 2 hydrogen bonds (weak) |
| Classification Tip Between Purines and Pyrimidines | Purines (A,G - 2 rings), Pyrimidines (C,T,U - 1 ring). A,G can be associated with ATP, an energy molecule that needs more to grab onto → 2 rings. And CTU has 3 letters, which form pyra and the U is like RNA, needs less → 1 ring. Or…pure silver. |
| Tip about Exam Question | there will be a multiple choice question pertaining to whether the model shows a purine or pyrimidine. |
| Complementary antiparallel strands (DNA Double Helix Properties): | Complementary antiparallel strands in a DNA double helix mean that the two strands run in opposite directions 5' to 3' and 3' to 5' and have a predictable base-pairing relationship: adenine (A) always pairs with thymine (T), and guanine (G) always pairs w |
| Major groove (DNA Double Helix Properties): | The major groove is about 22 Å wide and exposes the edges of base pairs, making it the primary site where proteins like transcription factors recognize and bind specific DNA sequences. |
| Minor groove (DNA Double Helix Properties): | The minor groove is narrower, about 12 Å wide, and also provides access to base-pair information, but it is less commonly used for sequence-specific protein interactions due to limited space. |
| Helix parameters (DNA Double Helix Properties): | 10 base pairs per complete turn, 3.4 nm per turn |
| 1 bp | 1 base pair |
| 1 kb | 1,000 base pairs (kilo) |
| 1 mb | 1,000,000 base pairs (mega) |
| E. coli Chromosome Scale: | ~5 mb (5,000,000 base pairs) |
| Physical length of E.coli Chromosome: | 1.7 mm when extended |
| E. Coli Cell size: | Only 1-2 μm long × 0.25-1.0 μm wide |
| E.Coli Packaging problem: | DNA must be compacted ~850-fold to fit in a cell. The solution is supercoiling and protein packaging. |
| E. coli K-12 Chromosome: | The best studied E. coli strain with a chromosome of 88% protein encoding genes, 1% tRNA and rRNA genes, 11% regulatory sequences, spacer regions, etc. |
| Operon: | a functional unit of DNA in bacteria that consists of a cluster of genes with related functions, all under the control of a single promoter. |
| Tip for Remembering E.Coli Size Units: | Nanometer (nm) < Micrometer (µm) < Millimeter (mm) < Centimeter (cm) < Decimeter (dm) < Meter (m) |
| Atomic Force Microscopy (AFM): | Needle goes across the sample and generates a third-dimensional picture. AFM uses a nanoscale probe to map the surface of cells or bacteria in 3D, revealing fine structural details without staining. |
| Simultaneous Phase Contrast & Fluorescence Microscopy: | This method lets you view live cell shapes (phase contrast) while also tracking specific molecules or proteins (fluorescence) in the same sample. |
| Plasmids (on slide 7): | The small circular DNA molecules highlighted by blue boxes and circles, which are independent genetic elements that can replicate separately from the main chromosome. |
| Chromosomes (on slide 7): | The large, light-colored oval structure represents the bacterial chromosome (nucleoid region). |
| Supercoiling Directions | DNA can be supertwisted in a positive or a negative direction. The enzyme gyrase introduces two negative supertwists per ATP hydrolysis. The function is that it facilitates DNA unwinding during replication and transcription. |
| How to Relax Supercoiling | Topoisomerase enzymes nick DNA to relieve supercoiling tension. Breaks one strand, rotate one end of the broken strand around the helix and seals it, +1 change. The gyrase change is -2. |
| Exception to Negative Supercoiling | Hyperthermophilic archaea use positive supercoiling to prevent unwinding at high temperatures. They have to have a “reverse gyrase” that works in the opposite direction. |
| The DNA Gyrase Mechanism | DNA gyrase binds to DNA, creates a temporary double-strand break, and passes another segment of the helix through it before resealing the break. This process introduces negative supercoils, compacting the bacterial chromosome. |
| Chromosome | the main genetic element in prokaryotic cells containing all the genes necessary for life (mostly circulator but can be linear & some species have more than one chromosome). |
| Plasmid | circular or linear double stranded DNA molecules that replicate separately from the chromosome and provide “extra” characteristics for the cell. |
| Genome | total complement of genetic elements in the cell. |
| oriC | The chromosomal origin of replication in E. coli, where DNA replication begins. |
| malKBM | A gene cluster in the maltose operon that encodes proteins for maltose transport and metabolism. |
| tolC | An outer membrane protein that forms a channel used in efflux pumps and secretion systems. |
| Plasmid Diversity | Different plasmids can exist in the same cell, Plasmids come in all sizes (<1 kb to >1 mb), Plasmids have different copy numbers (1 to >100), Often encode genes that allow survival under specialized conditions. |
| Example of Plasmids Surviving Under Specialized Conditions: | R Plasmids encoding resistance to antibiotics (R100 = mercury, sulfonamide, streptomycin, chloramphenicol, tetracycline). |
| Transposable Elements | mobile DNA sequences that can move within or between genomes, sometimes disrupting or regulating genes. Plasmids often contain transposable elements that can move from one location to another (i.e. Insertion Sequences, IS). |
| Semiconservative replication | Each new DNA molecule contains one original and one newly synthesized strand |
| Template requirement | New DNA synthesis requires existing DNA template |
| Directionality of DNA synthesis | DNA synthesis occurs only in 5' to 3' direction |
| How many DNA polymerase are there in E.coli? | There are five different DNA polymerases. DNA Polymerase III replicates the majority of DNA. DNA Polymerase I fills in gaps. Other polymerases are involved in repair mechanisms. |
| Important characteristics of DNA Polymerases: | Require a primer (DNA or RNA), require nucleotide triphosphates as the building blocks. The building blocks for this process are the four deoxynucleoside triphosphates (dNTPs) — dATP, dCTP, dGTP, and dTTP — for DNA, and four ribonucleoside triphosphates ( |
| Directionality of the DNA Polymerase | Synthesize DNA in the 5’ to 3’ direction, Catalyze nucleophilic attack of the 3’ hydroxyl to the 5’ phosphate releasing pyrophosphate, Proofread and have 3’ to 5’ exonuclease activity remove an added mismatched base (the enzyme “backs up”). |
| Where does DNA Polymerase Direct The Synthesis if there’s a mistake? | 3’ to 5’, opposite of normal, exonuclease activity removes mismatched bases. |
| Helicase | Unwinds DNA double helix at replication fork (requires ATP) |
| Single Strand Binding (SSB) protein: | Prevents single strands from re-annealing |
| Function of Primase: | Synthesizes RNA primers required for Pol III initiation |
| Function of Gyrase: | Relieves positive supercoiling ahead of replication fork |
| Function of DNA Ligase: | Seals gaps between Okazaki fragments |
| Leading & Lagging Strand Directions: | leading strand synthesized continuously in 5′ to 3′ direction, the lagging strand synthesized discontinuously in fragments, same direction. Two template strands are antiparallel, so one runs “backwards” relative to the movement of the replication fork. |
| Steps of Completing the Lagging Strand: | Pol III extends DNA to RNA primer but can't remove it (no exonuclease), Pol I removes RNA primer one nucleotide at a time (5'→3' exonuclease), Pol I fills gaps with DNA, Ligase seals nicks (requires 3'-OH + 5'-P), Results in continuous double-strand DNA |
| Mechanism of Lagging Strand: | DNA Polymerase I fills in gaps and removes the RNA primer, DNA ligase seals the separate DNA chains together |
| Bidirectional Replication in E.Coli Chromosome: | replication is initiated by DNaA protein at OriC (origin chromosome), there are two replication forks going in opposite directions, forms a theta structure (Greek letter), the two forks eventually meet each other, the two circular DNA are separated. |
| mRNA: | Messenger RNA - linear, short-lived, template for protein synthesis. Carries the genetic instructions copied from DNA in the nucleus to the ribosomes in the cytoplasm. |
| tRNA: | Transfer RNA - folded, stable, Decodes the mRNA sequence and delivers the corresponding amino acids to the ribosome. |
| rRNA: | Ribosomal RNA - folded, stable, structural/catalytic role in ribosomes |
| Reminders about rRNA: | RNA contains ribose not deoxyribose so it is less stable than DNA, RNA has uracil in place of thymine as one of the four bases, RNA is synthesized from nucleotide triphosphates (ATP, GTP, CTP, UTP). |
| Gene: | basic unit of genetic information, also defined as the nucleic acid sequence that codes for a polypeptide |
| Bacterial RNA Polymerase (only one): | does NOT require a primer (unlike DNA polymerase), Synthesizes 5' → 3' (same as DNA polymerase), Unwinds DNA by itself (doesn't need helicase), Reads template 3' → 5' |
| Bacterial RNA Polymerase Holoenzyme Subunits: | β, β', α₂, ω, σ |
| Bacterial RNA Polymerase Core enzyme (β, β', α₂, ω): | can synthesize an RNA chain from a DNA template but lacks specificity. It can transcribe non-specific sequences. Does not have a σ |
| Bacterial RNA Polymerase Sigma (σ) subunit: | Promoter recognition and binding. After initiation, σ dissociates. |
Created by:
smurtab