DNA Replication & Repair
โญ Enzymology of the Replisome
6 Parameters
Bacterial DNA Polymerases: Pol I vs Pol II vs Pol III vs Pol IV vs Pol V
Comparative enzymatic activities, exonuclease proofreading directions, processivity values, and cellular roles of all five E. coli DNA polymerases.
| Enzymatic Property | DNA Pol I (Kornberg) | DNA Pol II | DNA Pol III (Holoenzyme) | DNA Pol IV (DinB) & Pol V (UmuD'2C) |
|---|---|---|---|---|
| Primary Cellular Function | Primer removal & gap filling (Okazaki maturation) | DNA repair (restart stalled replication forks) | Primary genomic replicase (leading & lagging strand) | Translesion DNA synthesis (TLS / SOS error-prone bypass) |
| 5' โ 3' Polymerase Activity | Present | Present | Present | Present |
| 3' โ 5' Exonuclease (Proofreading) | Present | Present | Present (via ฮต subunit) | ABSENT (highly error-prone, no proofreading) |
| 5' โ 3' Exonuclease (Nick Translation) | PRESENT (Unique to Pol I) | Absent | Absent | Absent |
| Processivity (Nucleotides / binding) | Low (~15 โ 20 nt) | Moderate (~1,500 nt) | Extremely High (>500,000 nt via ฮฒ-clamp) | Very Low (~1 โ 10 nt) |
| Structural Composition | Single polypeptide (103 kDa; yields Klenow fragment) | Single polypeptide (90 kDa) | 10-subunit multimeric complex (core + clamp + clamp loader) | Multi-protein SOS response complex |
Exonuclease Activity Matrix
Pol I
Polymerase 5 3: Yes
Proofreading 3 5: Yes
Nick Translation 5 3: YES (Unique)
Pol II
Polymerase 5 3: Yes
Proofreading 3 5: Yes
Nick Translation 5 3: No
Pol III
Polymerase 5 3: Yes
Proofreading 3 5: Yes
Nick Translation 5 3: No
Pol IV / V
Polymerase 5 3: Yes
Proofreading 3 5: No
Nick Translation 5 3: No
Transcription & RNA Processing
โญ Gene Expression Machinery
6 Parameters
Prokaryotic vs Eukaryotic Transcription
Comprehensive comparative matrix contrasting RNA polymerases, promoter motifs, transcription factors, termination mechanisms, and post-transcriptional processing.
| Parameter | Prokaryotes (Bacteria) | Eukaryotes |
|---|---|---|
| RNA Polymerase Types | Single RNA Pol core (ฮฑ2ฮฒฮฒ'ฯ) + ฯ factor | Three distinct specialized RNA polymerases (Pol I, II, III) |
| Division of Labor (Pol I, II, III) | Single enzyme synthesizes all RNA classes | Pol I: 28S, 18S, 5.8S rRNA; Pol II: mRNA, snRNA, miRNA; Pol III: tRNA, 5S rRNA |
| Core Promoter Motifs | -10 Pribnow box (TATAAT) and -35 sequence (TTGACA) | TATA box (-25 to -30), Initiator (Inr), DPE (-30 to +30) |
| Promoter Recognition | Sigma factor (ฯ70, ฯ32, ฯ54) directly binds promoter | General Transcription Factors (TFIID/TBP, TFIIB, TFIIE, TFIIH) |
| Termination Mechanism | Intrinsic hairpin (GC-rich stem + poly-U) or Rho-dependent | Cleavage and polyadenylation signal (AAUAAA) coupled with Xrn2 torpedo |
| mRNA Structure & Processing | Polycistronic, uncapped, non-polyadenylated, no introns | Monocistronic, 5' 7-methylguanosine cap, 3' poly-A tail, spliced introns |
Polymerase & Transcript Organization
Prokaryote
Polymerases: 1 (Core + Sigma)
Promoter Element: -10 / -35 Box
Mrna Nature: Polycistronic, Unprocessed
Eukaryote
Polymerases: 3 (Pol I, II, III)
Promoter Element: TATA / Inr / DPE
Mrna Nature: Monocistronic, Spliced & Capped
Molecular Techniques
โญ Diagnostic Lab Techniques
5 Parameters
Molecular Blotting Techniques: Southern vs Northern vs Western vs Eastern
Standard diagnostic comparison of target macromolecule, separation gel, membrane type, and probing methods across all blotting modalities.
| Technique | Target Molecule | Gel Separation | Transfer Membrane | Probe / Detection Agent |
|---|---|---|---|---|
| Southern Blot (E.M. Southern, 1975) | DNA (Genomic fragments, RFLPs) | Agarose gel (denaturing alkaline buffer) | Nitrocellulose or Nylon | Radiolabeled (32P) or fluorescent DNA/RNA probe |
| Northern Blot | RNA (mRNA expression levels, transcript size) | Formaldehyde-agarose (denaturing RNA) | Nylon or Nitrocellulose | Radiolabeled cDNA or cRNA antisense probe |
| Western Blot (Immunoblot) | Proteins (size, post-translational abundance) | SDS-PAGE (polyacrylamide) | PVDF or Nitrocellulose | Primary monoclonal/polyclonal antibody + enzyme-conjugated secondary |
| Eastern Blot | Post-translational modifications (lipids, carbohydrates) | SDS-PAGE or 2D-PAGE | PVDF | Lectins, cholera toxin, or phospho-specific antibodies |
| Southwestern Blot | DNA-binding proteins (Transcription factors) | SDS-PAGE | Nitrocellulose | Double-stranded radiolabeled DNA oligonucleotides |
SNOW DROP Rule
Southern
Target: DNA
Probe: DNA/RNA Probe
Northern
Target: RNA
Probe: cDNA Probe
Western
Target: Protein
Probe: Specific Antibody
Eastern
Target: PTM (Glycan/Lipid)
Probe: Lectin / Toxins
