Cell Biology and Molecular Biology — Signalling, Trafficking, Replication, Expression and Repair
1. Eukaryotic cell structure, the cell cycle and growth control
A eukaryotic cell compartmentalises its work: the nucleus (with nuclear pores and the nucleolus, where rRNA is made), rough ER (secretory and membrane protein synthesis), smooth ER (lipid synthesis, detoxification, Ca²⁺ storage), Golgi (glycosylation and sorting), lysosomes (acid hydrolases), peroxisomes (β-oxidation of very long fatty acids, H₂O₂ breakdown by catalase), mitochondria and chloroplasts (with their own circular genomes, as endosymbiont descendants). The cytoskeleton has three polymers: actin microfilaments (about 7 nm), intermediate filaments (about 10 nm) and microtubules (about 25 nm, tubulin).
The cell cycle is G₁, S (DNA replication), G₂ and M, driven by cyclin–CDK complexes whose cyclins rise and fall: cyclin D–CDK4/6 in G₁, cyclin E–CDK2 at G₁/S, cyclin A–CDK2 in S, and cyclin B–CDK1 (maturation-promoting factor) at G₂/M. At the restriction point in G₁, CDK phosphorylation of the retinoblastoma protein Rb releases E2F, which switches on S-phase genes. Checkpoints stop the cycle for damage (p53 induces the CDK inhibitor p21 at G₁/S), incomplete replication (G₂/M) and unattached kinetochores (the spindle-assembly checkpoint holds back the anaphase-promoting complex). Cancer is growth control lost — gain-of-function in proto-oncogenes such as RAS and MYC, loss of function in tumour suppressors such as p53 and RB.
2. Cell–cell communication and signal transduction
Signals travel by blood (endocrine), to near neighbours (paracrine), back to the sender (autocrine) or by direct contact (juxtacrine, as when Delta on one cell activates Notch on the next and γ-secretase releases the Notch intracellular domain to the nucleus). Non-cell-autonomous signalling is the general case of one cell’s gene product deciding another cell’s fate — secreted morphogens such as Wnt, Hedgehog and BMP forming concentration gradients across a tissue, so that a mutant clone can change the behaviour of wild-type neighbours. Lipophilic hormones such as steroids cross the membrane and bind nuclear receptors that are themselves transcription factors; hydrophilic signals bind surface receptors and act through second messengers.
| Receptor | Transduction | Classic facts |
|---|---|---|
| G-protein-coupled (seven transmembrane helices) | Ligand makes Gα swap GDP for GTP. Gs activates adenylyl cyclase → cAMP → PKA; Gi inhibits it; Gq activates phospholipase C-β → IP₃ (Ca²⁺ release) and DAG (PKC) | Cholera toxin ADP-ribosylates Gαs and blocks its GTPase, so cAMP stays high; pertussis toxin ADP-ribosylates Gαi and uncouples it |
| Receptor tyrosine kinase | Ligand-induced dimerisation and trans-autophosphorylation; SH2 proteins dock; Grb2–SOS activates Ras, then Raf → MEK → ERK | Growth-factor receptors such as those for EGF and insulin; constitutive Ras is a common oncogenic mutation |
| Cytokine receptors | Associated JAK kinases phosphorylate STATs, which dimerise and enter the nucleus | Interferon and interleukin signalling |
| Nuclear receptors | Hormone–receptor complex binds hormone-response elements in DNA | Steroid, thyroid hormone and retinoic acid receptors |
3. Post-translational modification, protein trafficking, cell death and the extracellular matrix
- Post-translational modifications: phosphorylation of Ser, Thr and Tyr by kinases; N-linked glycosylation on Asn in the sequon Asn-X-Ser/Thr (X not Pro), begun co-translationally in the ER from a dolichol-linked precursor and trimmed in the Golgi; O-linked glycosylation on Ser/Thr in the Golgi; ubiquitination (a Lys48-linked polyubiquitin chain sends a protein to the 26S proteasome); acetylation, lipidation (myristoylation, prenylation, GPI anchors), disulfide formation and proteolytic processing.
- Protein trafficking: an N-terminal signal sequence is bound by the signal recognition particle, which docks the ribosome on the ER for co-translational translocation; KDEL returns escaped ER residents; mannose-6-phosphate tags lysosomal enzymes in the Golgi; a nuclear localisation signal is carried in by importins with the Ran-GTP gradient; mitochondrial precursors enter through TOM and TIM. Vesicle coats set direction — COPII from ER to Golgi, COPI back from Golgi to ER, clathrin from the plasma membrane and trans-Golgi network.
- Cell death: apoptosis is orderly and non-inflammatory. In the intrinsic pathway Bax and Bak permeabilise the outer mitochondrial membrane (Bcl-2 prevents it), cytochrome c is released and joins Apaf-1 in the apoptosome, which activates caspase-9; in the extrinsic pathway Fas ligand or TNF engages a death receptor and activates caspase-8. Both converge on executioner caspases 3 and 7. Necrosis is swelling and lysis with inflammation. Autophagy wraps cytoplasm and organelles in a double-membrane autophagosome (marked by lipidated LC3) that fuses with a lysosome; mTOR activity suppresses it, so starvation induces it.
- Extracellular matrix: collagen is a triple helix of Gly-X-Y repeats whose prolines and lysines are hydroxylated by enzymes that need ascorbate (hence scurvy without vitamin C); fibronectin binds integrins through its RGD motif; laminin builds the basal lamina; proteoglycans hydrate and cushion; integrins link the matrix to the actin cytoskeleton and signal in both directions.
4. Genes, chromosomes and DNA replication
A eukaryotic gene is exons and introns under a promoter with enhancers that may lie far away; a bacterial operon is several genes under one promoter. Chromatin is built from nucleosomes — about 147 bp of DNA wrapped 1.65 times round an octamer of two each of H2A, H2B, H3 and H4 — with H1 on the linker DNA outside the octamer, folded further into loops and the condensed mitotic chromosome. Heterochromatin is condensed and mostly silent; euchromatin is open. Centromeres assemble the kinetochore; telomeres are repeats capped by proteins and extended by telomerase, a reverse transcriptase carrying its own RNA template, which solves the end-replication problem of the lagging strand.
Replication is semi-conservative — Meselson and Stahl grew E. coli in ¹⁵N, moved it to ¹⁴N and separated DNA on a CsCl gradient: after one generation all DNA was hybrid, after two half hybrid and half light, and after n generations the hybrid fraction is 2/2ⁿ (0.25 after three). At each fork helicase (DnaB) unwinds, single-strand binding protein coats, gyrase relieves the supercoils ahead, primase lays RNA primers, DNA polymerase III extends 5′→3′ with 3′→5′ exonuclease proofreading, DNA polymerase I removes primers with its 5′→3′ exonuclease and fills the gaps, and ligase seals the nicks. The leading strand is continuous; the lagging strand is made as Okazaki fragments. Eukaryotes use many origins, with polymerase α–primase priming, ε mainly on the leading and δ mainly on the lagging strand.
5. Transcription, splicing, translation, their regulation, and non-coding RNA
| Feature | Bacteria | Eukaryotes |
|---|---|---|
| Polymerase | One core enzyme; σ⁷⁰ recognises the −35 (TTGACA) and −10 (TATAAT) boxes | Pol I: 28S, 18S and 5.8S rRNA; Pol II: mRNA, most snRNAs and miRNAs; Pol III: tRNA and 5S rRNA |
| Promoter recognition | σ factor directly | General transcription factors; TBP of TFIID binds the TATA box |
| Termination | Intrinsic (GC-rich hairpin then a U run) or Rho-dependent | Coupled to cleavage and polyadenylation for Pol II |
| Processing | mRNA is translated while still being made | 5′ 7-methylguanosine cap, splicing, 3′ poly(A) tail, export |
Splicing removes introns by two transesterifications in the spliceosome (U1, U2, U4/U6 and U5 snRNPs): the 2′-OH of the branch-point adenosine attacks the 5′ splice site (GU), forming a lariat, and the freed 3′-OH of the exon attacks the 3′ splice site (AG). Alternative splicing makes several proteins from one gene. Translation begins at AUG — positioned by the Shine–Dalgarno sequence pairing with 16S rRNA in bacteria and by cap scanning and the Kozak context in eukaryotes — on 70S (30S + 50S) or 80S (40S + 60S) ribosomes. EF-Tu delivers aminoacyl-tRNAs, the peptidyl transferase is the 23S rRNA, a ribozyme, EF-G translocates, and release factors read UAA, UAG and UGA. The code is degenerate (61 sense codons for 20 amino acids), with wobble at the third codon position. An open reading frame of 1503 nucleotides including its stop codon encodes 1503/3 − 1 = 500 amino acids.
Regulation of gene expression in eukaryotes works at chromatin (remodelling, histone marks, methylation), at transcription (activators on enhancers, repressors on silencers, bridged to the polymerase by Mediator), at RNA processing, export and stability, and at translation. Non-coding RNAs include rRNA, tRNA, snRNA, snoRNA, long non-coding RNAs such as XIST, and microRNAs: a primary transcript is cut by Drosha–DGCR8 in the nucleus into a hairpin pre-miRNA, exported by exportin-5, cut by Dicer into a duplex, and one strand is loaded into the RNA-induced silencing complex with an Argonaute protein. Imperfect pairing, usually in the 3′ UTR through the seed region, represses translation and destabilises the mRNA; perfect pairing, as with a designed siRNA in RNA interference, lets Argonaute cleave the target.
6. Mutations, mutagenesis, and DNA damage and repair
Point mutations are transitions (purine for purine, pyrimidine for pyrimidine) or transversions; by effect they are silent, missense or nonsense, and insertions or deletions not a multiple of three cause frameshifts. Mutagens act by recognisable chemistry: base analogues such as 5-bromouracil mispair in a tautomeric form and cause transitions; ethyl methanesulfonate ethylates guanine at O⁶, and O⁶-ethylguanine pairs with thymine, giving G·C → A·T transitions; nitrous acid deaminates cytosine to uracil; intercalating acridines such as proflavine cause frameshifts; UV light forms cyclobutane pyrimidine dimers. The Ames test screens chemicals for mutagenicity as reversion of His⁻ Salmonella, with a liver extract added to mimic metabolic activation.
| Pathway | Lesion | Mechanism and disease link |
|---|---|---|
| Direct reversal | Pyrimidine dimers; O⁶-alkylguanine | Photolyase uses visible light (photoreactivation); a methyltransferase takes the alkyl group onto itself |
| Base excision | Damaged or wrong bases such as uracil | A glycosylase removes the base, AP endonuclease cuts, polymerase and ligase fill in |
| Nucleotide excision | Bulky, helix-distorting lesions including UV dimers | UvrABC excises an oligonucleotide; defects cause xeroderma pigmentosum |
| Mismatch repair | Replication mispairs | MutS finds the mismatch, MutL recruits MutH, which nicks the unmethylated new strand at a hemimethylated GATC site (Dam methylation marks the parent); defects cause Lynch syndrome |
| Double-strand break repair | Broken chromosomes | Non-homologous end joining (fast, error-prone) or homologous recombination with a sister template (accurate; BRCA1/BRCA2) |
| SOS response | Heavy damage blocking replication | RecA triggers LexA cleavage; error-prone translesion polymerases IV and V bypass the damage |
Key takeaways
- Cyclin B–CDK1 drives G₂/M; Rb phosphorylation frees E2F at the restriction point; p53 halts the cycle through p21.
- Cholera toxin keeps Gαs on (cAMP high); Gq gives IP₃ and DAG; RTKs signal through Ras–MAPK; Notch is contact-dependent, morphogens are non-cell-autonomous.
- N-glycosylation at Asn-X-Ser/Thr starts in the ER; COPII carries ER to Golgi; intrinsic apoptosis runs cytochrome c → Apaf-1 → caspase-9, extrinsic runs death receptor → caspase-8.
- Replication is semi-conservative (hybrid fraction 2/2ⁿ); Pol III copies with proofreading, Pol I removes primers; Pol III of eukaryotes makes tRNA and 5S rRNA; peptidyl transferase is 23S rRNA.
- EMS gives G·C → A·T transitions, acridines frameshifts, UV pyrimidine dimers; NER fixes dimers (XP), mismatch repair reads hemimethylated GATC, NHEJ is error-prone and HR accurate.
Practice questions (13)
Attempt each one before opening the answer. Every explanation names the tempting wrong option as well as the right one, because that is where marks are lost.
Vesicles that carry newly made proteins from the endoplasmic reticulum to the Golgi apparatus are coated with
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Answer: A — COPII
COPII buds anterograde vesicles from ER exit sites. COPI mediates the reverse, retrograde traffic from the Golgi back to the ER and between Golgi cisternae, and clathrin coats vesicles from the plasma membrane and trans-Golgi network — the usual swap is COPI for COPII.Cholera toxin causes massive fluid loss from intestinal cells because it
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Answer: A — ADP-ribosylates Gαs, blocking its GTPase so adenylyl cyclase stays active
Cholera toxin’s A subunit transfers ADP-ribose to Gαs, which then cannot hydrolyse its GTP and stays switched on; cAMP rises, PKA opens the CFTR chloride channel, and water follows the chloride out. Modifying Gαi is what pertussis toxin does, which also raises cAMP but by a different route.N-linked glycosylation of a secretory protein occurs on
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Answer: A — asparagine in the sequence Asn-X-Ser/Thr, beginning in the ER
Oligosaccharyltransferase in the ER lumen transfers a preassembled dolichol-linked glycan onto the amide nitrogen of asparagine in the sequon Asn-X-Ser/Thr (X any residue but proline), and the Golgi later trims and extends it. O-linked glycans on serine or threonine are the ones added in the Golgi.Which of the following statements about the intrinsic (mitochondrial) pathway of apoptosis are correct?
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Answer: A — Cytochrome c is released from mitochondria into the cytosol.; B — Apaf-1 forms the apoptosome, which activates caspase-9.
In the intrinsic pathway Bax and Bak release cytochrome c, which binds Apaf-1 to build the apoptosome and activate caspase-9. Bcl-2 is anti-apoptotic — it prevents outer-membrane permeabilisation rather than promoting it — and Fas ligand starts the extrinsic pathway through caspase-8.E. coli grown for many generations in ¹⁵N medium is transferred to ¹⁴N medium. After three generations of replication, the fraction of DNA molecules that are ¹⁵N/¹⁴N hybrid is ______.
Numerical answer — type the value.
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Answer: 0.25
The two original heavy strands persist, each in one hybrid molecule, while the total number of molecules doubles every generation: after three generations there are 2³ = 8 molecules, 2 of them hybrid, so 2/8 = 0.25. Answering 0.125 counts one hybrid molecule instead of two.In eukaryotes, tRNAs and 5S rRNA are transcribed by
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Answer: A — RNA polymerase III
Pol III makes tRNA, 5S rRNA and a few small RNAs such as U6. Pol I makes only the large rRNA precursor that gives 28S, 18S and 5.8S rRNA in the nucleolus — the reason 5S is a trap, as the one rRNA not made by Pol I — and Pol II makes mRNA and most snRNAs and miRNAs.During methyl-directed mismatch repair in E. coli, the newly synthesised strand is identified because
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Answer: A — its GATC sites are transiently unmethylated, and MutH nicks it at a hemimethylated site
Dam methylase methylates the adenine in GATC only some time after replication, so for a short window the parent strand is methylated and the daughter is not. MutS binds the mismatch, MutL links it to MutH, and MutH nicks the unmethylated daughter strand, which is then excised and resynthesised. Uracil is a base-excision substrate and photolyase reverses UV dimers.A bacterial chromosome of 4.6 × 10⁶ bp is replicated bidirectionally from a single origin, each fork moving at 1000 nucleotides per second. The time needed to replicate the whole chromosome is ______ minutes (round off to one decimal place).
Numerical answer — type the value.
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Answer: 38.3
Two forks leave the origin in opposite directions and each copies half the circle, so the combined rate is 2 × 1000 = 2000 bp/s. Time = 4.6 × 10⁶/2000 = 2300 s = 38.3 min. Forgetting bidirectionality doubles it to 76.7 min; counting both strands as separate work would double it again.A small RNA loaded into the RNA-induced silencing complex pairs perfectly along its length with a target mRNA. The most likely outcome is
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Answer: A — endonucleolytic cleavage of the mRNA by Argonaute
Full complementarity lets the slicer activity of Argonaute cut the target between positions 10 and 11 of the guide, which is how siRNAs work in RNA interference. Seed-only pairing, typical of animal microRNAs in the 3′ UTR, instead represses translation and destabilises the message without slicing.Which of the following statements about the nucleosome are correct?
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Answer: A — About 147 bp of DNA is wrapped around the histone core.; B — The core octamer contains two each of H2A, H2B, H3 and H4.; D — Acetylation of histone lysines tends to loosen chromatin and favour transcription.
The core particle is about 147 bp on an octamer of two copies each of H2A, H2B, H3 and H4, and acetylation neutralises lysine’s positive charge, weakening its grip on DNA and recruiting activators. H1 is the linker histone: it binds outside the octamer where DNA enters and leaves, so it is not one of the eight.Ethyl methanesulfonate is used as a mutagen chiefly because it produces
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Answer: A — G·C → A·T transitions, through O⁶-ethylguanine pairing with thymine
EMS is an alkylating agent: ethylation of guanine at O⁶ makes a base that pairs with thymine, so after replication a G·C pair becomes A·T — overwhelmingly transitions, which is why EMS is the standard chemical mutagen in forward genetic screens. Intercalation is how acridines cause frameshifts, dimers come from UV, and deamination is the action of nitrous acid.An open reading frame is 1503 nucleotides long from the start codon to the end of the stop codon. The number of amino acids in the encoded polypeptide is ______.
Numerical answer — type the value.
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Answer: 500
1503/3 = 501 codons, of which the last is a stop codon that is read by a release factor and adds no amino acid, so 500 amino acids (counting the initiating methionine). Answering 501 counts the stop codon as if it encoded something.The cyclin–CDK complex that drives cells from G₂ into mitosis is
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Answer: A — cyclin B–CDK1
Cyclin B accumulates through S and G₂ and, bound to CDK1, forms maturation-promoting factor, whose activation by Cdc25 triggers nuclear-envelope breakdown and chromosome condensation; its destruction by the anaphase-promoting complex lets the cell leave mitosis. Cyclin D–CDK4 acts in G₁, and cyclin E–CDK2 and cyclin A–CDK2 at the G₁/S transition and in S.