Chapter 16: Gene Expression Flashcards
What are the five major levels of gene expression regulation?
- Epigenetic regulation (e.g., DNA methylation silencing tumor suppressor genes).
- Transcriptional regulation (e.g., transcription factors binding promoters).
- Post-transcriptional regulation (e.g., miRNAs cleaving target mRNAs).
- Translational regulation (e.g., phosphorylation of eIF2 blocking initiation).
- Post-translational regulation (e.g., ubiquitination tagging proteins for degradation).
Describe the structure and regulation of the lac operon.
Structure: Promoter (RNA polymerase binding site), operator (repressor binding site), and structural genes (lacZ, lacY, lacA).
Regulation:
- Repression: Without lactose, the repressor binds the operator, blocking transcription.
- Induction: Lactose (inducer) binds the repressor, freeing the operator.
- CAP activation: cAMP-CRP complex enhances transcription under low glucose.
How does attenuation regulate the trp operon?
High tryptophan: Ribosomes quickly translate the leader peptide, allowing mRNA to form a termination hairpin (attenuator), halting transcription.
Low tryptophan: Ribosomes stall, preventing terminator formation; transcription proceeds.
How does DNA methylation suppress gene expression?
Mechanism: Methylation of CpG islands recruits methyl-binding proteins (e.g., MeCP2), compacting chromatin and blocking transcription.
Example: Hypermethylation of BRCA1 in breast cancer silences this tumor suppressor.
List three types of histone modifications and their effects.
- Acetylation (e.g., H3K9ac): Opens chromatin, promoting transcription.
- Methylation (e.g., H3K27me3): Recruits repressors (e.g., Polycomb) to silence genes.
- Phosphorylation (e.g., H3S10ph): Triggers chromatin condensation during mitosis.
Explain X-chromosome inactivation.
Xist RNA: Coats one X chromosome, recruiting PRC2 to add H3K27me3 marks.
Result: Chromatin compaction and silencing (Barr body formation).
How do enhancers regulate distant genes?
Looping: Enhancers physically contact promoters via CTCF/cohesin-mediated DNA looping.
Coactivators: Transcription factors (e.g., p53) recruit mediator complexes to activate RNA Pol II.
How do steroid hormones regulate gene expression?
Mechanism: Hormones (e.g., estrogen) bind nuclear receptors → receptors dimerize → bind hormone response elements (HREs) → recruit histone acetyltransferases (HATs) → activate transcription.
How does alternative splicing increase protein diversity?
Mechanism: A single pre-mRNA is spliced in multiple ways (e.g., exon skipping, intron retention).
Example: Dscam in fruit flies generates >38,000 isoforms for neuronal connectivity.
Compare miRNA and siRNA.
miRNA: Endogenous; binds partially complementary sites in 3’UTR, blocking translation or degrading mRNA (e.g., let-7 in cell cycle control).
siRNA: Exogenous (e.g., viral RNA); binds perfectly complementary sites, cleaving mRNA via RNA-induced silencing complex (RISC).
How does the mTOR pathway regulate translation?
Activation: Nutrients/insulin → activate mTORC1 → phosphorylate 4E-BP → release eIF4E → enhance cap-dependent translation.
Inhibition: Starvation → inactive mTORC1 → 4E-BP binds eIF4E → block translation initiation.
Describe the ubiquitin-proteasome system.
Ubiquitination: E1 (activates), E2 (conjugates), E3 (ligase, e.g., SCF) tags substrate with polyubiquitin.
Degradation: Proteasome recognizes ubiquitin tags → unfolds and digests proteins (e.g., cyclin degradation in cell cycle).
How do p53 mutations contribute to cancer?
Normal role: Activates DNA repair or apoptosis genes (e.g., Bax) upon damage.
Mutation: Loss of function → unchecked cell division → genomic instability → tumorigenesis.
Name two epigenetic drugs used in cancer therapy.
- 5-Azacytidine: DNA methyltransferase inhibitor → reactivates silenced tumor suppressors.
- Vorinostat (SAHA): HDAC inhibitor → opens chromatin to restore pro-apoptotic gene expression.
Explain chromatin immunoprecipitation (ChIP).
Steps: Crosslink proteins-DNA → fragment chromatin → immunoprecipitate with target antibody (e.g., RNA Pol II) → sequence DNA to identify binding sites.
Application: Mapping transcription factor occupancy (e.g., NF-κB at inflammation genes).
Design an experiment to validate miRNA targeting a gene.
Prediction: Use tools like TargetScan to identify miRNA binding sites in the gene’s 3’UTR.
Luciferase assay: Clone the 3’UTR into a luciferase reporter plasmid → co-transfect with miRNA mimics → measure luciferase activity drop.
Functional validation: Overexpress miRNA → quantify mRNA (qRT-PCR) and protein (Western blot) levels of the target.
Mutagenesis: Disrupt miRNA binding sites in the 3’UTR → confirm loss of repression.
