Transcription (biology) Study Guide

📖 Core Concepts Transcription – copying a DNA segment into an RNA molecule; the primary way genes are expressed. Template (antisense) strand – the DNA strand read by RNA polymerase (3′→5′); its complement becomes RNA. Coding (sense) strand – has the same base order as the RNA transcript (U replaces T) and is shown when DNA sequences are written. RNA polymerase II (Pol II) CTD – repeats of the heptapeptide YSPTSPS; dynamic phosphorylation coordinates capping, splicing, and poly‑adenylation. Promoter – DNA region where the pre‑initiation complex (PIC) assembles; contains core elements (TATA, Inr, etc.) that define the transcription start site. Enhancer – distal regulatory DNA that can boost transcription up to 100‑fold by looping to the promoter; bound by tissue‑specific TFs and the Mediator complex. CpG island & DNA methylation – CpG‑rich promoter regions; 5‑mC addition represses transcription by recruiting MBD proteins and repressive chromatin remodelers. Abortive initiation & promoter escape – Pol II synthesizes short RNAs (10 nt) before a stable elongation complex forms; ATP hydrolysis by TFIIH drives escape. --- 📌 Must Remember Directionality: Template strand read 3′→5′ → RNA synthesized 5′→3′. Key Pol II factors: TFIIA, TFIIB, TFIID (TBP), TFIIE, TFIIF, TFIIH (phosphorylates CTD Ser‑5). Bacterial termination: Rho‑independent: GC hairpin + poly‑U tract. Rho‑dependent: Rho ATPase translocates on RNA to release the complex. Eukaryotic termination: Cleavage + poly‑A addition (torpedo model). Sigma factor – bacterial holoenzyme subunit that directs promoter recognition. Methyl‑binding domain (MBD) proteins (MeCP2, MBD1/2) bind 5‑mC and recruit repressive complexes. Reverse transcriptase – makes cDNA from RNA; telomerase is a specialized RT that uses its own RNA template. Key inhibitors: Rifampicin (bacterial Pol β‑subunit), Triptolide (XPB subunit of TFIIH). --- 🔄 Key Processes Initiation General TFs + Pol II bind promoter → closed complex. DNA unwinds (14 bp) → open complex (transcription bubble). First NTP binds; synthesis of the first phosphodiester bond. Promoter Escape Abortive cycles produce RNAs <10 nt. Once 10 nt RNA made, TFIIH hydrolyzes ATP → Pol II clears promoter → stable elongation complex. Elongation Pol II moves 3′→5′ along template, adds ribonucleotides to 5′ end of RNA. Nucleosomes act as barriers; TFIIS and nucleosome “breathing” aid passage. Termination (Bacteria) Rho‑independent: hairpin + poly‑U → RNA–DNA duplex destabilizes. Rho‑dependent: Rho binds rut site, hydrolyzes ATP, pulls RNA off. Termination (Eukaryotes) Poly‑A signal (AAUAAA) recognized → cleavage → exonuclease (Xrn2) degrades downstream RNA (torpedo). Enhancer‑Promoter Looping CTCF/YY1 dimer binds enhancer & promoter → DNA loop stabilized → Mediator transmits activator signal to Pol II. --- 🔍 Key Comparisons Template vs. Coding Strand Template: read by polymerase, 3′→5′. Coding: same sequence as RNA (U for T), displayed in databases. Rho‑independent vs. Rho‑dependent termination Hairpin‑polyU (intrinsic) vs. ATP‑dependent Rho translocation. Bacterial sigma factor vs. Eukaryotic TFIIH Sigma: promoter recognition (core promoter). TFIIH: phosphorylates Pol II CTD, ATP‑driven promoter escape. CpG‑rich vs. CpG‑poor promoters CpG‑rich: housekeeping genes, nucleosome‑free, often unmethylated. CpG‑poor: tissue‑specific, rely on TF binding sites, more nucleosome‑occupied. Rifampicin vs. Triptolide Rifampicin blocks bacterial Pol β‑subunit. Triptolide targets XPB (TFIIH) in eukaryotes. --- ⚠️ Common Misunderstandings “RNA polymerase reads the coding strand.” – It reads the antisense template strand; the coding strand is just a convenient reference. “All transcription termination is the same in bacteria and eukaryotes.” – Bacteria use hairpin/​Rho mechanisms; eukaryotes rely on cleavage/poly‑A and torpedo pathways. “Methylation always turns a gene off.” – Methylation in promoters represses transcription, but CpG methylation in gene bodies can have other regulatory roles. “Abortive transcripts are errors.” – They are a normal part of promoter escape; the polymerase discards them until a stable RNA ≥10 nt is made. --- 🧠 Mental Models / Intuition “Railroad track” model: DNA is the track; Pol II is a locomotive that must first uncouple (initiation), gain momentum (promoter escape), and then cruise (elongation) while navigating speed bumps (nucleosomes). “Looped telephone line” – Enhancer → Mediator → Pol II is like a telephone line that must be physically looped to transmit the signal; CTCF/YY1 are the clips that keep the line taut. “Hairpin brake” – In bacteria, the GC hairpin acts like a brake that forces the polymerase to stop when followed by a weak poly‑U “slippery” zone. --- 🚩 Exceptions & Edge Cases Promoter-proximal pausing: Even after escape, Pol II can pause 30‑60 nt downstream; NELF/DSIF complexes enforce this pause. CpG islands in active promoters can be partially methylated without full silencing, especially in cancer cells with heterogeneous methylation patterns. Rho‑independent termination can fail if the hairpin is destabilized (e.g., mutations reducing GC content). Some eukaryotic genes use alternative poly‑A signals, leading to alternative 3′ ends and isoforms. --- 📍 When to Use Which Identify transcription start site (TSS): Use the coding strand sequence; look for TATA box 30 bp upstream in promoters. Predict bacterial termination: Scan for GC‑rich hairpin followed by ≥4 U’s → likely Rho‑independent. Decide if a gene is epigenetically silenced: Check CpG island methylation status; presence of MBD binding suggests repression. Choose assay for expression profiling: RT‑PCR – precise quantification of a few targets. RNA‑Seq – genome‑wide expression, splice variants, fusions. Single‑cell RNA‑Seq – heterogeneity within a tissue. Select inhibitor for experimental blockade: Rifampicin – bacterial studies. Triptolide – mammalian Pol II studies (targets TFIIH). --- 👀 Patterns to Recognize Abortive → 10 nt → promoter escape – see a burst of short RNAs before stable transcripts. Hairpin‑polyU motif → bacterial intrinsic terminator. CTD Ser‑5 phosphorylation → early elongation & capping; later Ser‑2 phosphorylation → splicing & 3′ processing. eRNA production – bidirectional, short, non‑coding transcripts at active enhancers. Methyl‑CpG + MBD binding → compact chromatin, low transcriptional output. --- 🗂️ Exam Traps Distractor: “RNA polymerase reads the coding strand.” – Incorrect; it reads the template strand. Distractor: “All promoters contain a TATA box.” – Many CpG‑rich promoters lack a TATA box. Distractor: “Rifampicin inhibits eukaryotic transcription.” – It specifically targets bacterial RNA polymerase. Distractor: “Methylation always occurs at promoter CpG islands.” – DNA methylation also occurs in gene bodies and intergenic regions. Distractor: “Termination always follows a poly‑A signal in eukaryotes.” – Some transcripts (e.g., histone mRNAs) use a stem‑loop instead of poly‑A. ---