Gene expression Study Guide

📖 Core Concepts Gene expression – conversion of DNA‑encoded information into a functional product (protein or functional RNA). Gene classes – housekeeping (always on), facultative (on when needed), inducible (respond to signals). Transcription – synthesis of an RNA copy by RNA polymerase; RNA is complementary to the DNA template, U replaces T. RNA polymerases – Prokaryotes: one enzyme + sigma factor; Eukaryotes: Pol I (rRNA), Pol II (mRNA & most ncRNA), Pol III (tRNA, 5S rRNA). mRNA processing (eukaryotes) – 5′ cap, 3′ poly(A) tail, splicing (introns removed), export to cytoplasm. Alternative splicing – same pre‑mRNA can yield multiple mRNA isoforms, expanding proteome diversity. Translation – ribosome reads codons in the open‑reading frame, tRNAs bring amino acids, peptide chain elongates. Regulatory DNA elements – enhancers (activate), silencers (repress), insulators (block cross‑talk). Epigenetic marks – DNA methylation (CpG) usually represses promoters; histone acetylation opens chromatin. Post‑transcriptional control – 5′ cap & poly(A) tail stability, 3′ UTR‑bound miRNAs, RNA‑binding proteins. Post‑translational control – phosphorylation, ubiquitination, acetylation, methylation modify protein activity or half‑life. --- 📌 Must Remember Pol II CTD code: Ser‑5‑P → promoter clearance; Ser‑2‑P → elongation. Cap structure: 7‑methylguanosine (m⁷G) linked via a 5′‑5′ triphosphate bridge. Polyadenylation signal: AAUAAA → cleavage + 200 A residues. Spliceosome removes introns as lariat structures. Enhancer‑promoter looping is mediated by CTCF, YY1, Cohesin. DNA methylation: promoter CpG methylation ↓ transcription; gene‑body methylation can ↑ transcription. miRNA action: perfect complement → cleavage; partial complement → translational repression/decay. qRT‑PCR Ct value inversely proportional to initial RNA amount; lower Ct = higher expression. RNA‑seq detects splice variants, SNPs, novel transcripts; most comprehensive transcriptomic method. Western blot reveals protein size and post‑translational modifications via modification‑specific antibodies. --- 🔄 Key Processes Transcription Initiation (eukaryotes) TFs bind enhancer → Mediator recruitment → PIC assembly (TFIID → TFIIA → TFIIB → TFIIE → TFIIF → TFIIH). TFIIH helicase unwinds DNA; its kinase phosphorylates Pol II CTD (Ser‑5). mRNA Capping & Polyadenylation 5′ cap: guanylyltransferase adds GMP → methyltransferase creates m⁷G cap. Poly(A) tail: CPSF binds AAUAAA → endonuclease cleavage → poly(A) polymerase adds A’s → PAP binds PABP. Splicing & Alternative Splicing U1 snRNP binds 5′ splice site, U2 binds branch point, tri‑snRNP complex (U4/U5/U6) catalyzes intron removal → exon ligation. Translation Initiation (cap‑dependent) eIF4F (eIF4E‑cap, eIF4G‑scaffold, eIF4A‑helicase) binds 5′ cap → 40S subunit with eIF2‑GTP‑Met‑tRNA scans to AUG → 60S joins → elongation begins. miRNA Biogenesis Primary pri‑miRNA → Drosha/Pasha cleavage → pre‑miRNA hairpin → Exportin‑5 transport → Dicer cleavage → mature miRNA → RISC (Argonaute) loading. DNA Methylation/Demethylation DNMTs add CH₃ to 5‑C in CpG; TET enzymes oxidize 5‑mC → base excision repair removes it → unmethylated C restored. --- 🔍 Key Comparisons Facultative vs. Housekeeping genes – Facultative: expressed only under specific conditions; Housekeeping: constitutively expressed in all cells. Enhancer vs. Silencer – Enhancer ↑ transcription via activator TFs; Silencer ↓ transcription via repressor TFs. Pol I vs. Pol II vs. Pol III – Pol I: rRNA genes; Pol II: mRNA & most ncRNA; Pol III: tRNA, 5S rRNA, small ncRNA. Monocistronic vs. Polycistronic mRNA – Monocistronic (one ORF, typical eukaryote); Polycistronic (multiple ORFs, typical prokaryote). Cap‑dependent vs. IRES‑mediated translation – Cap‑dependent requires eIF4F and scanning; IRES allows direct ribosome recruitment under stress. --- ⚠️ Common Misunderstandings “All methylation represses transcription.” – CpG methylation in promoters represses, but gene‑body methylation can correlate with active transcription. “mRNA abundance equals protein level.” – Translation efficiency and protein stability can cause discordance. “Splicing only removes introns.” – It also creates exon‑exon junctions that affect export, nonsense‑mediated decay, and translation. “All miRNA binding leads to mRNA cleavage.” – In animals, most miRNAs cause translational repression rather than cleavage. “Enhancers act only locally.” – Enhancers can act megabases away via DNA looping. --- 🧠 Mental Models / Intuition “Factory assembly line” – DNA (blueprints) → transcription (copying blueprint) → processing (adding protective tags) → export (shipping) → translation (assembly). “Lock & key for regulation” – TFs are keys; promoters/enhancers are locks. Methylation adds a “padlock” that blocks the key. “Traffic lights on the CTD” – Ser‑5‑P = green for initiation; Ser‑2‑P = green for elongation; NELF/DSIF = red light (pause). “RNA as a zip code” – 5′ cap & poly(A) = passport for export; 3′ UTR motifs = address labels directing localization or decay. --- 🚩 Exceptions & Edge Cases Gene‑body methylation can enhance transcription, contrary to promoter methylation. Pol II transcription termination can be torpedo (Rat1/Xrn2 exonuclease) or pause‑release mechanisms. Alternative polyadenylation creates 3′ UTR isoforms that escape miRNA regulation. Some viral RNAs are capped in the nucleus (e.g., influenza) unlike most host mRNAs. eIF2α phosphorylation globally reduces translation but selectively up‑regulates ATF4 via upstream ORFs. --- 📍 When to Use Which Detecting splice variants → RNA‑seq (full‑length reads) rather than qRT‑PCR (requires specific primers). Quantifying a single low‑abundance transcript → RT‑qPCR (high sensitivity) over Northern blot. Measuring protein modifications → Western blot with phospho‑ or ubiquitin‑specific antibodies. Assessing genome‑wide DNA methylation → Bisulfite sequencing (not listed but implied). Studying enhancer activity → eRNA detection (RNA‑seq) or chromatin conformation capture (Hi‑C). --- 👀 Patterns to Recognize AAUAAA downstream of a cleavage site → polyadenylation signal. GC‑rich promoter region + SP1 binding sites → housekeeping gene. CpG island + unmethylated → active promoter; methylated → silenced. Multiple exons flanked by GU‑AG → classic splice junction. High 3′ UTR length + many AU‑rich elements → rapid mRNA turnover. --- 🗂️ Exam Traps Choosing the wrong polymerase – Pol II transcribes most protein‑coding genes; Pol I does not. Assuming all miRNA binding leads to cleavage – most animal miRNAs repress translation without cleavage. Confusing enhancer vs. promoter sequences – enhancers are distal, can act in either orientation, and often lack TATA boxes. Interpreting a strong Northern blot band as high protein level – mRNA abundance does not guarantee high protein; consider translation efficiency. Mixing up 5′ cap vs. 3′ poly(A) functions – cap protects 5′ end and aids export; poly(A) protects 3′ end and enhances translation. ---