Molecular Biology Expert

DNA Structure & Replication

DNA double helix:
  Antiparallel strands: 5′→3′ and 3′→5′
  Base pairing: A-T (2 H-bonds), G-C (3 H-bonds)
  B-DNA: right-handed, 10 bp/turn, 3.4 nm pitch
  Major groove: wide, protein binding
  Minor groove: narrow, some drug binding

Chromosomal organization:
  Prokaryotes: circular chromosome + plasmids, nucleoid region
  Eukaryotes: linear chromosomes, histones, chromatin
  Histone octamer: 2×(H2A,H2B,H3,H4) + H1 linker
  Nucleosome: 147 bp DNA wrapped around octamer
  30 nm fiber → loops → domains → chromosome

DNA replication (eukaryotic):
  Semi-conservative: each daughter has one old + one new strand
  Bidirectional from multiple origins (ori) simultaneously
  Origin recognition complex (ORC) marks origins
  MCM helicase unwinds DNA (loaded during G1)

Key enzymes:
  Helicase (MCM): unwinds double helix
  Primase: synthesizes short RNA primers
  DNA Pol α: extends primers (low fidelity)
  DNA Pol δ/ε: main replicative polymerases (high fidelity, 3′→5′ proofreading)
  DNA Pol γ: mitochondrial replication
  RNase H / FEN1: removes RNA primers
  DNA ligase: seals nicks (uses ATP in eukaryotes)
  Topoisomerase I: relieves torsional stress (nicks)
  Topoisomerase II: relieves torsional stress (double strand breaks)
  PCNA: sliding clamp, processivity factor
  RPA: single-strand DNA binding protein

Leading vs lagging strand:
  Leading: continuous synthesis 5′→3′ toward replication fork
  Lagging: discontinuous Okazaki fragments (100-200 nt in eukaryotes)
  Okazaki fragments joined by RNase H + FEN1 + DNA Pol δ + Ligase

Telomeres and telomerase:
  Telomeres: TTAGGG repeats (humans), protect chromosome ends
  End replication problem: lagging strand cannot replicate very end
  Telomerase: reverse transcriptase with RNA template, extends 3′ end
  Somatic cells: telomeres shorten with each division → senescence/apoptosis
  Stem cells, cancer cells: telomerase active → immortality

Transcription

def transcription_overview():
    return {
        'Prokaryotic transcription': {
            'RNA Pol': 'Single core enzyme (α₂ββ\'ω) + σ factor',
            'σ factor': 'Recognizes -10 and -35 promoter elements',
            'σ⁷⁰': 'Default housekeeping σ factor (E. coli)',
            'initiation': 'σ binds promoter → open complex → RNA synthesis',
            'elongation': 'σ dissociates, ~50 nt/sec',
            'termination': {
                'Rho-independent': 'Stem-loop structure + U-rich sequence',
                'Rho-dependent': 'Rho helicase catches up to RNA Pol → dissociation'
            }
        },
        'Eukaryotic RNA Polymerases': {
            'RNA Pol I': 'rRNA (28S, 18S, 5.8S) — nucleolus',
            'RNA Pol II': 'mRNA, most snRNA, miRNA — main enzyme',
            'RNA Pol III': '5S rRNA, tRNA, small RNAs'
        },
        'Eukaryotic mRNA transcription': {
            'Core promoter': 'TATA box (~-25), Inr, BRE, DPE',
            'TFIID': 'TBP + TAFs, binds TATA box first',
            'GTFs': 'TFIIA, B, D, E, F, H assemble preinitiation complex',
            'CTD': 'C-terminal domain of Pol II: YSPTSPS heptapeptide repeats',
            'CTD phosphorylation': 'Ser5 (initiation/capping) → Ser2 (elongation)',
            'enhancers': 'Can be kb away, looping to promoter via Mediator'
        }
    }

RNA Processing

5′ capping:
  Addition of 7-methylguanosine cap co-transcriptionally (after ~25 nt)
  Cap structure: m⁷G-ppp-N1 (inverted)
  Functions: protection from 5′ exonucleases, translation initiation, splicing

3′ polyadenylation:
  Cleavage at poly(A) signal (AAUAAA + downstream GU-rich)
  Poly(A) polymerase adds 200-250 adenosines
  Functions: export, stability, translation efficiency

Pre-mRNA splicing:
  Introns removed, exons joined
  Splice sites: GU at 5′ end, AG at 3′ end (GT-AG rule)
  Branch point: A residue ~20-50 nt upstream of 3′ splice site
  
  Mechanism (two transesterifications):
  Step 1: 2′-OH of branch A attacks 5′ splice site → lariat intermediate
  Step 2: 3′-OH of upstream exon attacks 3′ splice site → joined exons
  
  Spliceosome: snRNPs (U1, U2, U4, U5, U6) + proteins
  U1 snRNA: base pairs with 5′ splice site
  U2 snRNA: base pairs with branch point
  U4/U6/U5 tri-snRNP: catalytic complex

Alternative splicing:
  Exon skipping, intron retention, alternative 5′ or 3′ sites
  ~95% of human genes alternatively spliced
  Examples: tropomyosin (muscle type specific), Dscam (insect, 38,016 isoforms)

Non-coding RNAs:
  miRNA: ~22 nt, RISC complex, translational repression or mRNA degradation
  siRNA: ~21 nt, RNAi pathway, perfect complementarity → mRNA cleavage
  lncRNA: >200 nt, chromatin regulation, scaffolding
  circRNA: covalently circular, sponge for miRNAs
  piRNA: silence transposons in germline
  snRNA: spliceosome components
  snoRNA: guide rRNA modification

Translation

Genetic code:
  61 sense codons + 3 stop (UAA, UAG, UGA)
  Redundant/degenerate: multiple codons for same AA
  Wobble: third position less stringent (Crick, 1966)
  Nearly universal (mitochondria have slight variations)
  Start codon: AUG (Met)

Ribosomes:
  Prokaryote: 70S = 30S + 50S
    30S: 16S rRNA + ~20 proteins (decoding)
    50S: 23S + 5S rRNA + ~30 proteins (peptidyl transferase)
  Eukaryote: 80S = 40S + 60S
    40S: 18S rRNA (decoding)
    60S: 28S + 5.8S + 5S rRNA (peptidyl transferase)

tRNA structure:
  ~73-93 nt, cloverleaf secondary structure, L-shaped tertiary
  Anticodon loop: positions 34-36 (anticodon)
  Acceptor stem: 5′ end + 3′ CCA-OH (amino acid attachment)
  Aminoacyl-tRNA synthetase: charges tRNA with cognate amino acid

Eukaryotic translation initiation:
  1. eIF4E binds m⁷G cap
  2. eIF4A (helicase), eIF4G scaffold
  3. 43S PIC (40S + Met-tRNA + eIFs) recruited
  4. Scanning 5′→3′ for AUG in Kozak context (GCCRCCAUGG)
  5. 60S subunit joins → 80S initiation complex
  Cap-independent: IRES (internal ribosome entry site)

Elongation:
  A site: aminoacyl-tRNA entry (eEF1A·GTP)
  P site: peptidyl-tRNA (donor)
  E site: exit (deacylated tRNA)
  Peptidyl transfer: peptide transferred from P-site to A-site tRNA
  Translocation: eEF2·GTP moves ribosome 3 nt (one codon)

Termination:
  Stop codon in A site: eRF1 (recognizes all 3 stops) + eRF3·GTP
  Peptide hydrolysis: eRF1 triggers release
  Ribosome recycling: ABCE1 + eIF3 disassemble complex

Post-translational modifications:
  Phosphorylation, glycosylation, ubiquitination, SUMOylation
  Proteolytic processing, disulfide bonds, lipidation
  Protein folding: Hsp70, Hsp90, chaperonins (GroEL/GroES)

Gene Regulation

def gene_regulation():
    return {
        'Prokaryotic operons': {
            'Lac operon': {
                'genes':        'lacZ (β-gal), lacY (permease), lacA (transacetylase)',
                'repressor':    'LacI binds operator → blocks transcription',
                'inducer':      'Allolactose (from lactose) → binds LacI → conformational change → dissociates',
                'CAP/CRP':      'Glucose starvation → high cAMP → CAP-cAMP activates promoter',
                'regulation':   'Two inputs: lactose present AND glucose absent for max expression'
            },
            'Trp operon': {
                'repressor':    'TrpR inactive alone; binds tryptophan (corepressor) → active',
                'attenuation':  'Ribosome stalling near Trp codons controls termination',
                'logic':        'High Trp → full repression + attenuation'
            }
        },
        'Eukaryotic transcription regulation': {
            'Enhancers':    'Distal regulatory elements (can be >1 Mb away)',
            'Silencers':    'Reduce transcription',
            'Insulators':   'Block enhancer-promoter communication',
            'TF binding':   'Transcription factors: DBD + activation/repression domain',
            'Coactivators': 'Mediator complex, HATs (histone acetyltransferases)',
            'Corepressors': 'HDACs (histone deacetylases)',
            'Combinatorial':'Multiple TFs combinatorially control gene expression'
        },
        'Post-transcriptional regulation': {
            'mRNA stability':   'AU-rich elements (ARE) in 3\'UTR → destabilization',
            'miRNA':            'Seed sequence (~7 nt) targets 3\'UTR → repression',
            'RNA editing':      'A-to-I (adenosine deaminase), C-to-U editing',
            'Translation':      'uORFs, IRES, RNA structure regulate translation'
        }
    }

Epigenetics

DNA methylation:
  CpG methylation (5-methylcytosine, 5mC) in mammals
  DNMT1: maintenance methylation (copies pattern after replication)
  DNMT3A/3B: de novo methylation
  TET enzymes: oxidize 5mC → 5hmC → demethylation pathway
  CpG islands: ~1 kb regions, high CpG, often unmethylated at active promoters
  Methylated promoters: gene silencing (heterochromatin, X-inactivation, imprinting)

Histone modifications:
  H3K4me3: active promoters
  H3K27ac: active enhancers
  H3K36me3: actively transcribed gene bodies
  H3K27me3: Polycomb repression (gene silencing)
  H3K9me3: constitutive heterochromatin (repeat elements)
  H3K4me1: poised/active enhancers
  H4K16ac: active transcription, DNA damage response

Chromatin remodeling complexes:
  SWI/SNF (BAF): slide/eject nucleosomes (activation)
  NuRD: deacetylation + nucleosome remodeling (repression)
  ISWI: space nucleosomes evenly
  INO80: DNA repair, replication

Polycomb/Trithorax system:
  PRC2: H3K27 methyltransferase (EZH2 catalytic)
  PRC1: H2A ubiquitination, chromatin compaction
  Trithorax (MLL): H3K4 methylation, active gene maintenance

Genomic imprinting:
  Allele-specific methylation (paternal or maternal)
  ~80 imprinted genes in humans
  IGF2/H19, Prader-Willi/Angelman syndrome loci

X-inactivation:
  XIST lncRNA: coats inactive X chromosome
  Xist recruits PRC2, SPEN → H3K27me3 → silencing
  Random in somatic cells, established in early development

Molecular Techniques

def molecular_techniques():
    return {
        'PCR (Polymerase Chain Reaction)': {
            'principle':    'Exponential amplification of target DNA',
            'steps':        '94°C denature → 50-65°C anneal → 72°C extend (repeat 25-35×)',
            'components':   'Template, primers (F+R), Taq polymerase, dNTPs, buffer, Mg²⁺',
            'product':      '2ⁿ copies per cycle (n = cycle number)',
            'variants': {
                'RT-PCR':   'mRNA → cDNA (reverse transcriptase) → PCR',
                'qPCR':     'Real-time quantification using fluorescent dye/probe',
                'ddPCR':    'Digital PCR: absolute quantification in droplets',
                'Nested':   'Two rounds with inner primers (high sensitivity)',
                'LAMP':     'Loop-mediated isothermal amplification (no thermocycler)'
            }
        },
        'DNA Sequencing': {
            'Sanger':       'Dideoxy chain termination; gold standard for validation',
            'NGS':          'Next-gen: Illumina (SBS), Ion Torrent (pH), 454 (pyrosequencing)',
            'Long read':    'PacBio (SMRT), Oxford Nanopore (ionic current)',
            'Applications': 'WGS, WES, RNA-seq, ChIP-seq, ATAC-seq, scRNA-seq'
        },
        'Cloning': {
            'Restriction':  'Cut with restriction enzymes, ligate into vector',
            'Gibson':       'Overlap extension + exonuclease chewback (seamless)',
            'TOPO':         'Topoisomerase I-mediated (blunt or T-overhang)',
            'Gateway':      'Site-specific recombination (attB × attP)'
        },
        'Blotting': {
            'Southern':     'DNA: gel electrophoresis + transfer + probe hybridization',
            'Northern':     'RNA: detect specific transcripts',
            'Western':      'Protein: SDS-PAGE + transfer + antibody detection',
            'EMSA':         'Electrophoretic mobility shift: DNA-protein binding'
        },
        'Fluorescence': {
            'FISH':         'Fluorescence in situ hybridization: chromosome location',
            'IF':           'Immunofluorescence: protein localization in cells',
            'FRET':         'Förster resonance energy transfer: protein interactions',
            'FRAP':         'Fluorescence recovery: protein dynamics'
        },
        'Protein techniques': {
            'Co-IP':        'Co-immunoprecipitation: protein-protein interactions',
            'ChIP':         'Chromatin immunoprecipitation: DNA-protein interactions',
            'Y2H':          'Yeast two-hybrid: protein interaction screen',
            'BRET/BIFC':    'Bioluminescence/bimolecular fluorescence complementation'
        }
    }

CRISPR-Cas9 & Genome Editing

CRISPR-Cas9 mechanism:
  Guide RNA (gRNA = crRNA + tracrRNA or sgRNA)
  sgRNA: 20 nt spacer + scaffold
  Cas9: endonuclease, makes blunt cut 3 bp upstream of PAM
  PAM: 5′-NGG-3′ (SpCas9), protospacer adjacent motif
  Cas9 recognizes PAM → unwinds DNA → checks complementarity → cleaves both strands

Repair pathways:
  NHEJ (Non-Homologous End Joining): fast, error-prone → indels → knockout
  HDR (Homology-Directed Repair): template-based → precise edit (slow, needs template)
  
Applications:
  Gene knockout: NHEJ-induced frameshift
  Gene knockin: HDR with donor template
  CRISPRi: dCas9-KRAB → gene repression (no cutting)
  CRISPRa: dCas9-VPR → gene activation
  Base editing: dCas9-deaminase → C→T or A→G (no DSB)
  Prime editing: pegRNA + RT → any edit without DSB or HDR
  CRISPR screens: genome-wide sgRNA library → phenotype

Other nucleases:
  TALENs: TALE DNA-binding domain + FokI nuclease
  ZFNs: Zinc finger + FokI (older, harder to design)
  Meganucleases: natural endonucleases with long recognition sites

Delivery:
  Plasmid, viral (AAV, lentivirus), RNP (Cas9 protein + sgRNA), mRNA + sgRNA
  In vivo: LNP (lipid nanoparticles) — used in sickle cell therapy (Casgevy)

Omics Technologies

def omics_overview():
    return {
        'RNA-seq': {
            'measures':     'Transcriptome: all mRNA levels',
            'workflow':     'RNA → cDNA library → sequencing → alignment → counting → DEG',
            'tools':        'STAR (alignment), DESeq2/edgeR (differential expression)',
            'applications': 'Gene expression, alternative splicing, fusion genes'
        },
        'scRNA-seq': {
            'measures':     'Transcriptome of individual cells',
            'platforms':    '10x Genomics (droplet), Smart-seq2 (full length)',
            'analysis':     'Clustering → cell type identification',
            'tools':        'Seurat, Scanpy',
            'applications': 'Cell atlas, development, heterogeneity'
        },
        'ChIP-seq': {
            'measures':     'Genome-wide TF binding or histone modification',
            'workflow':     'Crosslink → ChIP → DNA → library → sequencing → peak calling',
            'tools':        'MACS2 (peak calling), DiffBind',
            'applications': 'Regulatory elements, enhancer maps, TF binding'
        },
        'ATAC-seq': {
            'measures':     'Open chromatin (accessible regions)',
            'principle':    'Tn5 transposase cuts and tags accessible DNA',
            'applications': 'Enhancer activity, TF footprinting, nucleosome positioning'
        },
        'Hi-C': {
            'measures':     '3D genome organization',
            'principle':    'Crosslink → restriction digest → proximity ligation → sequencing',
            'features':     'TADs, loops, compartments (A/B)',
            'tools':        'HiCExplorer, Juicer'
        },
        'Proteomics': {
            'mass spec':    'LC-MS/MS: trypsin digest → peptide separation → MS/MS ID',
            'quantification': 'Label-free, TMT, SILAC',
            'interactome':  'AP-MS, BioID (proximity labeling)'
        }
    }

Common Pitfalls

Related Skills

• cell-biology-expert: Cellular context of molecular processes
• genetics-expert: Inheritance and mutation
• biochemistry-expert: Protein structure and metabolism
• genomics-expert: Large-scale genome analysis
• bioinformatics-expert: Computational analysis of molecular data
• immunology-expert: Molecular immunology