Molecular Biology — Genes, Replication & Expression — USMLE Step 1 Notes

Chromatin structure

• Nucleosome: DNA wound around a histone octamer (2 each of H2A, H2B, H3, H4); H1 is the linker that packs nucleosomes together. DNA is negatively charged and binds positively charged (lysine/arginine-rich) histones.
• Heterochromatin: densely packed, transcriptionally inactive, stains dark; it is highly methylated and hypoacetylated (e.g. the Barr body/inactive X).
• Euchromatin: loosely packed, transcriptionally active ('Eu = true/expressed'), acetylated.

Epigenetics: DNA methylation & histone modification

• DNA methylation: addition of methyl groups (usually to cytosine in CpG islands) that silences genes; it underlies genomic imprinting and X-inactivation. Aberrant hypermethylation of tumour-suppressor promoters silences them in cancer.
• Pharmacology (methylation): hypomethylating agents azacitidine and decitabine reactivate silenced genes and are used in myelodysplastic syndrome/AML.
• Histone acetylation: histone acetyltransferases add acetyl groups, neutralising the positive charge, loosening chromatin and activating transcription.
• Histone deacetylation: histone deacetylases (HDACs) remove acetyl groups, tightening chromatin and silencing transcription. HDAC inhibitors (vorinostat, romidepsin) reopen chromatin and are used in cutaneous T-cell lymphoma.
• Histone methylation: context-dependent — it usually silences (e.g. H3K9, H3K27) but can activate (H3K4) depending on the residue.

Purine salvage disorders

• Adenosine deaminase (ADA) deficiency: deoxyadenosine and dATP accumulate and are toxic to lymphocytes → autosomal recessive severe combined immunodeficiency (SCID).
• Lesch-Nyhan syndrome: X-linked HGPRT deficiency blocks purine salvage → excess purine degradation, hyperuricaemia, gout, intellectual disability and self-mutilation. Treat hyperuricaemia with allopurinol.

DNA replication & its enzymes

• Origin & direction: replication is semi-conservative and bidirectional; the leading strand is continuous, the lagging strand is made in Okazaki fragments.
• Helicase unwinds the double helix at the fork (defective in Bloom syndrome); single-strand binding proteins stabilise the strands; topoisomerase/gyrase relieves supercoiling (the target of fluoroquinolones in bacteria).
• Primase lays down an RNA primer; DNA polymerase extends 5' to 3' (with 3'-to-5' proofreading exonuclease activity).
• DNA ligase seals the nicks between Okazaki fragments (deficient in Bloom syndrome).
• Telomerase is a reverse transcriptase that adds repeats to chromosome ends; it is usually silent in somatic cells (telomere shortening/ageing) but reactivated in most cancers.

DNA repair (pathophysiology & pharmacology)

• Nucleotide excision repair removes bulky UV-induced pyrimidine dimers; its failure causes xeroderma pigmentosum (severe photosensitivity and skin cancers).
• Mismatch repair corrects replication errors; germline defects cause Lynch syndrome (HNPCC) — colorectal and endometrial cancer.
• Base excision repair fixes single damaged bases (glycosylase, AP endonuclease).
• Homologous recombination repairs double-strand breaks; BRCA1/2 defects impair it, raising breast/ovarian cancer risk — and make tumours sensitive to PARP inhibitors (synthetic lethality). Ataxia-telangiectasia (ATM defect) also impairs double-strand-break repair.

Mutations in DNA

• Silent: a base change with the same amino acid (degeneracy of the code) — usually harmless.
• Missense: one amino acid replaced by another (e.g. sickle cell); a 'conservative' missense swaps a similar amino acid.
• Nonsense: creates a premature stop codon → truncated protein.
• Frameshift: insertion/deletion not a multiple of three shifts the reading frame (e.g. Duchenne, Tay-Sachs).
• Trinucleotide-repeat expansion: Huntington, Fragile X, myotonic dystrophy, Friedreich ataxia (anticipation).

Gene regulation: the lac operon

• A classic prokaryotic inducible operon. With low glucose and lactose present, transcription is maximal: lactose (as allolactose) inactivates the repressor (removing the brake), and low glucose raises cAMP, which binds CAP to boost RNA polymerase binding (the accelerator).
• Illustrates dual control — negative (repressor) and positive (CAP/cAMP) regulation.

Transcription & RNA

• RNA polymerases (eukaryotic): Pol I makes rRNA, Pol II makes mRNA (and is inhibited by alpha-amanitin from Amanita mushrooms), Pol III makes tRNA.
• mRNA processing: a 5' cap (7-methylguanosine), a 3' poly-A tail, and splicing out of introns (intervening, 'stay IN the nucleus') while joining exons (EXpressed) by the spliceosome. Alternative splicing lets one gene make several proteins; anti-spliceosomal (anti-U1 RNP) autoantibodies feature in mixed connective tissue disease.
• RNA types: mRNA carries the code (largest, most heterogeneous); tRNA brings amino acids and reads codons via its anticodon (has a CCA 3' end and unusual bases); rRNA (most abundant) is the catalytic core of the ribosome (a ribozyme).

Protein synthesis (translation) & pharmacology

• Steps: initiation (small ribosomal subunit + initiator tRNA find the start codon AUG), elongation (peptidyl transferase in the large subunit forms peptide bonds; needs GTP), and termination at a stop codon (UAA/UAG/UGA) with release factors.
• Ribosomes: eukaryotic 40S+60S = 80S; prokaryotic 30S+50S = 70S — the difference lets antibiotics selectively target bacteria.
• Antibiotics (30S): aminoglycosides (block initiation, misread) and tetracyclines (block tRNA entry). 50S: chloramphenicol (peptidyl transferase), macrolides/clindamycin/linezolid (translocation/initiation). ('Buy AT 30, CCEL at 50'.)
• Diphtheria and Pseudomonas exotoxin A inactivate eukaryotic EF-2 (ADP-ribosylation), halting translation.

Chaperones & protein folding

• Chaperones (e.g. heat-shock proteins like Hsp70/Hsp90) help nascent proteins fold correctly and refold stressed proteins, preventing aggregation.
• Signal recognition particle (SRP) targets ribosomes making secretory/membrane proteins to the rough endoplasmic reticulum.
• Pathophysiology: misfolded-protein aggregation underlies many diseases (e.g. prion disease, and the retained mutant Z protein in alpha-1 antitrypsin deficiency); Hsp90 inhibition is an anticancer strategy.

High-yield revision notes; verify against a primary source before clinical use.