{"format":"molecular-machines.cards","version":1,"updated":"2026-10-01","decks":[{"machine":"rna-polymerase","name":"RNA polymerase II","count":28,"url":"/learn/cards/rna-polymerase.json"}],"cards":[{"id":"rna-polymerase-makes-mrna","machine":"rna-polymerase","kind":"cloze","prompt":"RNA polymerase II copies a DNA gene into {{messenger RNA}}, the molecule that ribosomes read.","answer":"messenger RNA","explanation":"It works one base at a time: it pairs the template strand with a growing RNA chain.","section":"summary","topic":"purpose","sources":["machine:summary","machine:tagline"],"tags":["purpose","mrna"],"difficulty":1,"url":"/machines/rna-polymerase#summary","cites":[{"source":"machine:summary","machine":"rna-polymerase","label":"Summary","section":"summary"},{"source":"machine:tagline","machine":"rna-polymerase","label":"Summary","section":"summary"}]},{"id":"rna-polymerase-energy-source","machine":"rna-polymerase","kind":"qa","prompt":"What pays for each nucleotide that RNA polymerase II adds to the RNA?","answer":"The incoming nucleoside triphosphate itself: the energy of its triphosphate.","explanation":"The new bond takes the energy of the triphosphate, and pyrophosphate is split off and leaves.","section":"summary","topic":"purpose","sources":["machine:energy","step:The bond forms"],"tags":["energy","ntp"],"difficulty":1,"url":"/machines/rna-polymerase#summary","cites":[{"source":"machine:energy","machine":"rna-polymerase","label":"Summary","section":"summary"},{"source":"step:The bond forms","machine":"rna-polymerase","label":"Step: The bond forms","section":"mechanism"}]},{"id":"rna-polymerase-cleft-subunits","machine":"rna-polymerase","kind":"cloze","prompt":"In RNA polymerase II, the two largest subunits, {{Rpb1 and Rpb2}}, build the DNA cleft and the active site.","answer":"Rpb1 and Rpb2","explanation":"Rpb1 also carries the bridge helix, the trigger loop, the clamp core and metal A; Rpb2 forms the second wall of the cleft.","section":"summary","topic":"parts","sources":["stat:Subunits","component:Rpb1","component:Rpb2","ref:bushnell2003"],"tags":["subunits","active-site"],"difficulty":2,"url":"/machines/rna-polymerase#summary","cites":[{"source":"stat:Subunits","machine":"rna-polymerase","label":"Key number: Subunits","section":"summary"},{"source":"component:Rpb1","machine":"rna-polymerase","label":"Part: Rpb1","section":"summary"},{"source":"component:Rpb2","machine":"rna-polymerase","label":"Part: Rpb2","section":"summary"},{"source":"ref:bushnell2003","machine":"rna-polymerase","label":"Bushnell and Kornberg, PNAS 2003","section":"sources","anchor":"ref-bushnell2003","href":"https://doi.org/10.1073/pnas.1130601100"}]},{"id":"rna-polymerase-rpb4-rpb7-role","machine":"rna-polymerase","kind":"qa","prompt":"Which part of RNA polymerase II sits next to the RNA exit groove and binds the emerging transcript?","answer":"The Rpb4-Rpb7 stalk.","explanation":"Crystal structures also show Rpb7 wedging between the clamp and the linker to the tail domain, which locks the clamp closed.","section":"summary","topic":"parts","sources":["component:Rpb4-Rpb7 stalk","evidence:reconstituted-pol2-twelve-subunits","evidence:complete-pol2-twelve-subunits"],"tags":["subunits","rna-exit"],"difficulty":2,"url":"/machines/rna-polymerase#summary","cites":[{"source":"component:Rpb4-Rpb7 stalk","machine":"rna-polymerase","label":"Part: Rpb4-Rpb7 stalk","section":"summary"},{"source":"evidence:reconstituted-pol2-twelve-subunits","machine":"rna-polymerase","label":"Subunit count of complete RNA polymerase II, independent structure (Armache KJ 2003)","section":"evidence","anchor":"ev-reconstituted-pol2-twelve-subunits"},{"source":"evidence:complete-pol2-twelve-subunits","machine":"rna-polymerase","label":"Subunit count of complete RNA polymerase II (Bushnell DA 2003)","section":"evidence","anchor":"ev-complete-pol2-twelve-subunits"}]},{"id":"rna-polymerase-subunit-count","machine":"rna-polymerase","kind":"cloze","prompt":"Complete RNA polymerase II has {{12}} subunits: a ten-subunit core plus the Rpb4-Rpb7 pair.","answer":"12","explanation":"Bushnell and Kornberg tagged Rpb4 so every purified enzyme carried the pair, then solved the whole complex at 4.1 Å.","section":"summary","topic":"numbers","sources":["stat:Subunits","evidence:complete-pol2-twelve-subunits","ref:bushnell2003"],"tags":["subunits"],"difficulty":1,"url":"/machines/rna-polymerase#summary","cites":[{"source":"stat:Subunits","machine":"rna-polymerase","label":"Key number: Subunits","section":"summary"},{"source":"evidence:complete-pol2-twelve-subunits","machine":"rna-polymerase","label":"Subunit count of complete RNA polymerase II (Bushnell DA 2003)","section":"evidence","anchor":"ev-complete-pol2-twelve-subunits"},{"source":"ref:bushnell2003","machine":"rna-polymerase","label":"Bushnell and Kornberg, PNAS 2003","section":"sources","anchor":"ref-bushnell2003","href":"https://doi.org/10.1073/pnas.1130601100"}]},{"id":"rna-polymerase-hybrid-length","machine":"rna-polymerase","kind":"cloze","prompt":"Inside transcribing RNA polymerase II, the DNA-RNA hybrid is {{9}} base pairs long.","answer":"9","explanation":"The hybrid runs from the active site at nearly a right angle to the entering DNA (yeast Pol II crystal structure, 3.3 Å).","section":"summary","topic":"numbers","sources":["stat:DNA-RNA hybrid length","evidence:dna-rna-hybrid-nine-bp","ref:gnatt2001"],"tags":["hybrid","structure"],"difficulty":1,"url":"/machines/rna-polymerase#summary","cites":[{"source":"stat:DNA-RNA hybrid length","machine":"rna-polymerase","label":"Key number: DNA-RNA hybrid length","section":"summary"},{"source":"evidence:dna-rna-hybrid-nine-bp","machine":"rna-polymerase","label":"Length of the DNA-RNA hybrid in the elongation complex (Gnatt AL 2001)","section":"evidence","anchor":"ev-dna-rna-hybrid-nine-bp"},{"source":"ref:gnatt2001","machine":"rna-polymerase","label":"Gnatt et al.","section":"sources","anchor":"ref-gnatt2001","href":"https://doi.org/10.1126/science.1059495"}]},{"id":"rna-polymerase-antibiotic-target","machine":"rna-polymerase","kind":"qa","prompt":"Which state of RNA polymerase is an explicit target for new antibiotics, according to structures with streptolydigin?","answer":"The preinsertion state: the NTP held before the trigger loop folds over it.","explanation":"Streptolydigin holds the trigger loop away, so the nucleotide stays in an inactive preinsertion position. This inhibitor-design idea is lab-scale.","section":"summary","topic":"debate","sources":["frontier:Structure-guided inhibitor design","evolution:Shared two-step substrate path","ref:vassylyev2007b"],"tags":["trigger-loop","drugs"],"difficulty":3,"url":"/machines/rna-polymerase#summary","cites":[{"source":"frontier:Structure-guided inhibitor design","machine":"rna-polymerase","label":"Open question: Structure-guided inhibitor design","section":"summary"},{"source":"evolution:Shared two-step substrate path","machine":"rna-polymerase","label":"Shared two-step substrate path","section":"summary"},{"source":"ref:vassylyev2007b","machine":"rna-polymerase","label":"Vassylyev et al.","section":"sources","anchor":"ref-vassylyev2007b","href":"https://doi.org/10.1038/nature05931"}]},{"id":"rna-polymerase-t7-private-channel","machine":"rna-polymerase","kind":"qa","prompt":"Why does phage T7 RNA polymerase give a private expression channel in an E. coli cell?","answer":"It transcribes only T7 promoters, so it copies only the chosen gene.","explanation":"One subunit does the whole job and needs no factors. It is still the standard tool for recombinant protein work.","section":"summary","topic":"debate","sources":["frontier:Orthogonal expression with T7 RNA polymerase","species:T7 RNA polymerase","ref:studier1986"],"tags":["t7","biotech"],"difficulty":2,"url":"/machines/rna-polymerase#summary","cites":[{"source":"frontier:Orthogonal expression with T7 RNA polymerase","machine":"rna-polymerase","label":"Open question: Orthogonal expression with T7 RNA polymerase","section":"summary"},{"source":"species:T7 RNA polymerase","machine":"rna-polymerase","label":"T7 RNA polymerase","section":"summary"},{"source":"ref:studier1986","machine":"rna-polymerase","label":"Studier and Moffatt, Journal of Molecular Biology 1986","section":"sources","anchor":"ref-studier1986","href":"https://doi.org/10.1016/0022-2836(86)90385-2"}]},{"id":"rna-polymerase-post-translocation","machine":"rna-polymerase","kind":"qa","prompt":"In which translocation state can an NTP pair in the addition site of RNA polymerase II?","answer":"The post-translocation state, with template base i+1 facing the empty addition site.","explanation":"Until an NTP binds and holds it forward, the enzyme can still slide back to the pre-translocation state by thermal motion.","section":"mechanism","topic":"cycle","sources":["step:The addition site is open"],"tags":["translocation","ntp"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The addition site is open","machine":"rna-polymerase","label":"Step: The addition site is open","section":"mechanism"}]},{"id":"rna-polymerase-trigger-loop-folds","machine":"rna-polymerase","kind":"qa","prompt":"What does the trigger loop of RNA polymerase II do when a matching NTP pairs in the addition site?","answer":"It folds into a hairpin under the NTP, touches its base, sugar and phosphates, and seals the active site.","explanation":"Leu1081 touches the base, Gln1078 reaches the 3′-OH through Asn479, and His1085 binds the β-phosphate.","section":"mechanism","topic":"cycle","sources":["step:The trigger loop folds","mechanism:Substrate selection","ref:wang2006"],"tags":["trigger-loop"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The trigger loop folds","machine":"rna-polymerase","label":"Step: The trigger loop folds","section":"mechanism"},{"source":"mechanism:Substrate selection","machine":"rna-polymerase","label":"Step: Substrate selection","section":"mechanism"},{"source":"ref:wang2006","machine":"rna-polymerase","label":"Wang et al.","section":"sources","anchor":"ref-wang2006","href":"https://doi.org/10.1016/j.cell.2006.11.023"}]},{"id":"rna-polymerase-trigger-loop-coupling","machine":"rna-polymerase","kind":"qa","prompt":"Why does the trigger loop link NTP recognition to catalysis in RNA polymerase II?","answer":"The same loop that checks the NTP places His1085 where it may trigger the reaction.","explanation":"The loop closes over a correct NTP, so recognising the NTP and catalysis are coupled.","section":"mechanism","topic":"cycle","sources":["step:The trigger loop folds","evidence:wang2006-trigger-loop-contacts","ref:wang2006"],"tags":["trigger-loop","fidelity"],"difficulty":3,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The trigger loop folds","machine":"rna-polymerase","label":"Step: The trigger loop folds","section":"mechanism"},{"source":"evidence:wang2006-trigger-loop-contacts","machine":"rna-polymerase","label":"Trigger-loop contacts with the NTP in the addition site (Wang D 2006)","section":"evidence","anchor":"ev-wang2006-trigger-loop-contacts"},{"source":"ref:wang2006","machine":"rna-polymerase","label":"Wang et al.","section":"sources","anchor":"ref-wang2006","href":"https://doi.org/10.1016/j.cell.2006.11.023"}]},{"id":"rna-polymerase-two-metals-roles","machine":"rna-polymerase","kind":"qa","prompt":"Of the two Mg2+ ions in the active site of RNA polymerase II, which stays and which comes and goes each cycle?","answer":"Metal A stays bound to Rpb1 aspartates; metal B comes in with the NTP and leaves with the pyrophosphate.","explanation":"Metal A sits by the RNA 3′-OH and metal B holds the triphosphate, about 4 Å apart.","section":"mechanism","topic":"cycle","sources":["step:Pyrophosphate leaves","step:The NTP pairs with the template","stat:Active-site metals","step:The bond forms","evidence:metal-site-spacing","ref:wang2006"],"tags":["metals","catalysis"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:Pyrophosphate leaves","machine":"rna-polymerase","label":"Step: Pyrophosphate leaves","section":"mechanism"},{"source":"step:The NTP pairs with the template","machine":"rna-polymerase","label":"Step: The NTP pairs with the template","section":"mechanism"},{"source":"stat:Active-site metals","machine":"rna-polymerase","label":"Key number: Active-site metals","section":"summary"},{"source":"step:The bond forms","machine":"rna-polymerase","label":"Step: The bond forms","section":"mechanism"},{"source":"evidence:metal-site-spacing","machine":"rna-polymerase","label":"Spacing of the Mg2+ sites in transcribing Pol II (Wang D 2006)","section":"evidence","anchor":"ev-metal-site-spacing"},{"source":"ref:wang2006","machine":"rna-polymerase","label":"Wang et al.","section":"sources","anchor":"ref-wang2006","href":"https://doi.org/10.1016/j.cell.2006.11.023"}]},{"id":"rna-polymerase-loop-opens-before-ppi","machine":"rna-polymerase","kind":"qa","prompt":"Why must the trigger loop of RNA polymerase II open before pyrophosphate can leave?","answer":"The closed loop blocks the way out and holds the pyrophosphate through His1085.","explanation":"Only the fully open loop lets pyrophosphate move back to the entry site and out through the pore (Yi et al. 2026 preprint; Li et al. 2026 agrees).","section":"mechanism","topic":"cycle","sources":["step:The trigger loop opens","evidence:yi2026-ppi-bound-closed-loop","evidence:li2026-postcatalysis-ppi","ref:yi2026"],"tags":["trigger-loop","pyrophosphate"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The trigger loop opens","machine":"rna-polymerase","label":"Step: The trigger loop opens","section":"mechanism"},{"source":"evidence:yi2026-ppi-bound-closed-loop","machine":"rna-polymerase","label":"Pyrophosphate in the product state (Yi G 2026)","section":"evidence","anchor":"ev-yi2026-ppi-bound-closed-loop"},{"source":"evidence:li2026-postcatalysis-ppi","machine":"rna-polymerase","label":"Pyrophosphate after the bond forms, peer-reviewed structure (Li Q 2026)","section":"evidence","anchor":"ev-li2026-postcatalysis-ppi"},{"source":"ref:yi2026","machine":"rna-polymerase","label":"Yi et al.","section":"sources","href":"https://doi.org/10.64898/2026.06.04.730248"}]},{"id":"rna-polymerase-brownian-ratchet","machine":"rna-polymerase","kind":"cloze","prompt":"In the Brownian ratchet model, RNA polymerase slides back and forth along the DNA by thermal motion, and {{an incoming NTP}} catches it in the forward position.","answer":"an incoming NTP","explanation":"Each cycle moves the DNA and RNA one base pair; the trigger loop swings between open, wedged and closed positions as part of this motion.","section":"mechanism","topic":"cycle","sources":["step:DNA and RNA move one base pair","mechanism:Translocation","ref:abbondanzieri2005"],"tags":["translocation","ratchet"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:DNA and RNA move one base pair","machine":"rna-polymerase","label":"Step: DNA and RNA move one base pair","section":"mechanism"},{"source":"mechanism:Translocation","machine":"rna-polymerase","label":"Step: Translocation","section":"mechanism"},{"source":"ref:abbondanzieri2005","machine":"rna-polymerase","label":"Abbondanzieri et al.","section":"sources","href":"https://doi.org/10.1038/nature04268"}]},{"id":"rna-polymerase-proofreading-backtrack","machine":"rna-polymerase","kind":"qa","prompt":"How does RNA polymerase II remove a wrong base it has just added?","answer":"It backtracks one position, and the same active site cuts off the RNA end that holds the error.","explanation":"The wrong base first frays away from the template and the enzyme pauses. DNA polymerases, in contrast, use a separate nuclease site.","section":"mechanism","topic":"cycle","sources":["mechanism:Proofreading","stat:Proofreading","evolution:Proofreading in one active site","ref:sydow2009mc"],"tags":["proofreading","fidelity"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"mechanism:Proofreading","machine":"rna-polymerase","label":"Step: Proofreading","section":"mechanism"},{"source":"stat:Proofreading","machine":"rna-polymerase","label":"Key number: Proofreading","section":"summary"},{"source":"evolution:Proofreading in one active site","machine":"rna-polymerase","label":"Proofreading in one active site","section":"summary"},{"source":"ref:sydow2009mc","machine":"rna-polymerase","label":"Sydow et al.","section":"sources","anchor":"ref-sydow2009mc","href":"https://doi.org/10.1016/j.molcel.2009.06.002"}]},{"id":"rna-polymerase-tfiis-rescue","machine":"rna-polymerase","kind":"cloze","prompt":"The factor {{TFIIS}} rescues backtracked RNA polymerase II by helping it cut its RNA.","answer":"TFIIS","explanation":"In a backtracked enzyme the RNA end runs into the pore and traps the trigger loop; TFIIS reaches into the active site, pushes the RNA out and helps cut it.","section":"mechanism","topic":"parts","sources":["step:The next base loads","evidence:backtracked-rna-site","ref:cheung2011"],"tags":["backtracking","proofreading"],"difficulty":2,"url":"/machines/rna-polymerase#mechanism","cites":[{"source":"step:The next base loads","machine":"rna-polymerase","label":"Step: The next base loads","section":"mechanism"},{"source":"evidence:backtracked-rna-site","machine":"rna-polymerase","label":"Backtracked RNA bound in arrested Pol II (Cheung AC 2011)","section":"evidence","anchor":"ev-backtracked-rna-site"},{"source":"ref:cheung2011","machine":"rna-polymerase","label":"Cheung and Cramer, Nature 2011","section":"sources","anchor":"ref-cheung2011","href":"https://doi.org/10.1038/nature09785"}]},{"id":"rna-polymerase-same-genome-different-cells","machine":"rna-polymerase","kind":"qa","prompt":"A skin cell and a nerve cell carry the same genome. What makes them differ, in terms of what RNA polymerase II copies?","answer":"Which genes (pages) they read.","explanation":"Every cell with a nucleus holds the full set of instructions; RNA polymerase II copies only the genes that cell needs.","section":"story","topic":"purpose","sources":["stop:body","analogy:A scribe copying one page from a huge library"],"tags":["purpose","genome"],"difficulty":1,"url":"/machines/rna-polymerase#story","cites":[{"source":"stop:body","machine":"rna-polymerase","label":"Big picture: You, a library","section":"story"},{"source":"analogy:A scribe copying one page from a huge library","machine":"rna-polymerase","label":"Analogy: A scribe copying one page from a huge library","section":"story"}]},{"id":"rna-polymerase-dystrophin-time","machine":"rna-polymerase","kind":"cloze","prompt":"One RNA polymerase II takes about {{16 hours}} to copy the human dystrophin gene once.","answer":"16 hours","explanation":"Dystrophin is at least 2,300 kb long; muscle cell cultures copied about 1,770 kb in about 12 h (about 2.4 kb per minute), and the authors extrapolated.","section":"story","topic":"numbers","sources":["fact:V9","stop:gene","ref:tennyson1995"],"tags":["speed","genes"],"difficulty":2,"url":"/machines/rna-polymerase#story","cites":[{"source":"fact:V9","machine":"rna-polymerase","label":"Time to copy the dystrophin gene once: 16 h","section":"story"},{"source":"stop:gene","machine":"rna-polymerase","label":"Big picture: One long gene","section":"story"},{"source":"ref:tennyson1995","machine":"rna-polymerase","label":"Tennyson 1995","section":"sources","href":"https://doi.org/10.1038/ng0295-184"}]},{"id":"rna-polymerase-copies-per-hela","machine":"rna-polymerase","kind":"qa","prompt":"About how many RNA polymerase II molecules does one HeLa cell hold?","answer":"About 320,000.","explanation":"About 65,000 (roughly 20%) copy a gene at one time, but the source cites that share from earlier work rather than measuring it.","section":"story","topic":"numbers","sources":["fact:V12","ref:kimura1999"],"tags":["copy-number"],"difficulty":2,"url":"/machines/rna-polymerase#story","cites":[{"source":"fact:V12","machine":"rna-polymerase","label":"RNA polymerase II molecules in one HeLa cell: 320,000","section":"story"},{"source":"ref:kimura1999","machine":"rna-polymerase","label":"Kimura 1999","section":"sources","href":"https://doi.org/10.1128/mcb.19.8.5383"}]},{"id":"rna-polymerase-ribosome-link","machine":"rna-polymerase","kind":"cloze","prompt":"In a fast-growing yeast cell, {{half}} of all RNA polymerase II transcription serves the genes for ribosomal proteins.","answer":"half","explanation":"Ribosomes read the messenger RNA that RNA polymerase II writes.","section":"story","topic":"purpose","sources":["link:ribosome","ref:warner1999"],"tags":["ribosome","links"],"difficulty":2,"url":"/machines/rna-polymerase#story","cites":[{"source":"link:ribosome","machine":"rna-polymerase","label":"Link to Ribosome","section":"story"},{"source":"ref:warner1999","machine":"rna-polymerase","label":"Warner 1999","section":"sources","href":"https://doi.org/10.1016/s0968-0004(99)01460-7"}]},{"id":"rna-polymerase-train-analogy-breaks","machine":"rna-polymerase","kind":"qa","prompt":"Where does the \"train on a track\" analogy for RNA polymerase II break down?","answer":"Pol II pauses, backs up to fix errors, and speeds up and slows down along a gene.","explanation":"The analogy gets one thing right: Pol II runs along the gene in one direction.","section":"story","topic":"cycle","sources":["analogy:A train on a track","stat:Elongation rate, genome-wide","ref:jonkers2014"],"tags":["analogy","speed"],"difficulty":2,"url":"/machines/rna-polymerase#story","cites":[{"source":"analogy:A train on a track","machine":"rna-polymerase","label":"Analogy: A train on a track","section":"story"},{"source":"stat:Elongation rate, genome-wide","machine":"rna-polymerase","label":"Key number: Elongation rate, genome-wide","section":"summary"},{"source":"ref:jonkers2014","machine":"rna-polymerase","label":"Jonkers et al.","section":"sources","anchor":"ref-jonkers2014","href":"https://doi.org/10.7554/elife.02407"}]},{"id":"rna-polymerase-rate-long-genes","machine":"rna-polymerase","kind":"cloze","prompt":"Over long human genes, RNA polymerase II copies about {{3.8 kb per minute}}, about 63 nucleotides per second.","answer":"3.8 kb per minute","explanation":"Measured in human Tet-21 cells: 3.79 ± 0.26 kb per minute over 15 gene regions. That is about 16 ms per nucleotide.","section":"evidence","topic":"numbers","sources":["evidence:elongation-rate-long-human-genes","stat:Elongation rate in human cells","step:The addition site is open","ref:singh2009"],"tags":["speed"],"difficulty":1,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:elongation-rate-long-human-genes","machine":"rna-polymerase","label":"Pol II elongation rate over long endogenous human genes (Singh J 2009)","section":"evidence","anchor":"ev-elongation-rate-long-human-genes"},{"source":"stat:Elongation rate in human cells","machine":"rna-polymerase","label":"Key number: Elongation rate in human cells","section":"summary"},{"source":"step:The addition site is open","machine":"rna-polymerase","label":"Step: The addition site is open","section":"mechanism"},{"source":"ref:singh2009","machine":"rna-polymerase","label":"Singh and Padgett, Nature Structural and Molecular Biology 2009","section":"sources","anchor":"ref-singh2009","href":"https://doi.org/10.1038/nsmb.1666"}]},{"id":"rna-polymerase-rate-method","machine":"rna-polymerase","kind":"qa","prompt":"How did Singh and Padgett measure the speed of RNA polymerase II over long human genes?","answer":"They blocked new transcription with DRB, washed it out, and timed when new pre-mRNA reached exon-intron junctions far apart in the same gene.","explanation":"The delay between two junctions, read by RT-PCR, gives the speed; it was about the same over long introns and exon-rich stretches.","section":"evidence","topic":"numbers","sources":["evidence:elongation-rate-long-human-genes","ref:singh2009"],"tags":["speed","method"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:elongation-rate-long-human-genes","machine":"rna-polymerase","label":"Pol II elongation rate over long endogenous human genes (Singh J 2009)","section":"evidence","anchor":"ev-elongation-rate-long-human-genes"},{"source":"ref:singh2009","machine":"rna-polymerase","label":"Singh and Padgett, Nature Structural and Molecular Biology 2009","section":"sources","anchor":"ref-singh2009","href":"https://doi.org/10.1038/nsmb.1666"}]},{"id":"rna-polymerase-error-rate","machine":"rna-polymerase","kind":"qa","prompt":"About what transcription error rate per base was measured across all mRNA of the worm Caenorhabditis elegans?","answer":"About 4 x 10^-6 per base.","explanation":"That is about four mistakes per million letters (4.1 × 10^-6 pooled over three strains).","section":"evidence","topic":"numbers","sources":["evidence:transcription-error-rate","stat:Transcription error rate","fact:V11","ref:gout2013"],"tags":["fidelity"],"difficulty":2,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:transcription-error-rate","machine":"rna-polymerase","label":"Base-substitution error rate in mRNA in vivo (Gout JF 2013)","section":"evidence","anchor":"ev-transcription-error-rate"},{"source":"stat:Transcription error rate","machine":"rna-polymerase","label":"Key number: Transcription error rate","section":"summary"},{"source":"fact:V11","machine":"rna-polymerase","label":"Transcription errors: 4 × 10^-6 per letter","section":"story"},{"source":"ref:gout2013","machine":"rna-polymerase","label":"Gout et al.","section":"sources","anchor":"ref-gout2013","href":"https://doi.org/10.1073/pnas.1309843110"}]},{"id":"rna-polymerase-error-rate-method","machine":"rna-polymerase","kind":"qa","prompt":"How did Gout and colleagues tell real transcription errors apart from errors made while copying and sequencing the RNA?","answer":"They tagged each RNA fragment, copied it three times, and counted a change only if every copy carried it.","explanation":"Requiring the change in every copy removes errors made during copying and sequencing.","section":"evidence","topic":"numbers","sources":["evidence:transcription-error-rate","ref:gout2013"],"tags":["fidelity","method"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:transcription-error-rate","machine":"rna-polymerase","label":"Base-substitution error rate in mRNA in vivo (Gout JF 2013)","section":"evidence","anchor":"ev-transcription-error-rate"},{"source":"ref:gout2013","machine":"rna-polymerase","label":"Gout et al.","section":"sources","anchor":"ref-gout2013","href":"https://doi.org/10.1073/pnas.1309843110"}]},{"id":"rna-polymerase-mismatch-slows-next","machine":"rna-polymerase","kind":"qa","prompt":"According to Thomas and colleagues, what delay, more than the slow addition of the wrong base itself, lets RNA polymerase II tell right from wrong?","answer":"The next nucleotide is added at least 15- to 20-fold more slowly after a mismatched RNA end.","explanation":"The pause gives the enzyme time to back up and cut out the error. Measured with Pol II complexes from human nuclear extract.","section":"evidence","topic":"cycle","sources":["evidence:mismatch-slows-next-addition","evidence:misincorporation-slower-than-correct","ref:thomas1998"],"tags":["fidelity","proofreading"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:mismatch-slows-next-addition","machine":"rna-polymerase","label":"Extension rate after a mismatched RNA 3' end (Thomas MJ 1998)","section":"evidence","anchor":"ev-mismatch-slows-next-addition"},{"source":"evidence:misincorporation-slower-than-correct","machine":"rna-polymerase","label":"Rate of correct versus incorrect nucleotide incorporation (Thomas MJ 1998)","section":"evidence","anchor":"ev-misincorporation-slower-than-correct"},{"source":"ref:thomas1998","machine":"rna-polymerase","label":"Thomas et al.","section":"sources","anchor":"ref-thomas1998","href":"https://doi.org/10.1016/S0092-8674(00)81191-5"}]},{"id":"rna-polymerase-deoxy-ntp-chemistry","machine":"rna-polymerase","kind":"qa","prompt":"How did Wang and colleagues show that RNA polymerase II rejects 2′-deoxy NTPs mainly at the chemical step, not at binding?","answer":"Deoxy NTPs were added at least 400-fold more slowly, while their KM values were much closer to normal.","explanation":"A deoxy NTP still binds the addition site, so binding alone cannot tell it from an RNA building block; trigger-loop closure and the chemistry do.","section":"evidence","topic":"cycle","sources":["evidence:deoxy-ntp-discrimination","step:The NTP pairs with the template","ref:wang2006"],"tags":["fidelity","ntp"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:deoxy-ntp-discrimination","machine":"rna-polymerase","label":"Rate of 2'-deoxy NTP addition (Wang D 2006)","section":"evidence","anchor":"ev-deoxy-ntp-discrimination"},{"source":"step:The NTP pairs with the template","machine":"rna-polymerase","label":"Step: The NTP pairs with the template","section":"mechanism"},{"source":"ref:wang2006","machine":"rna-polymerase","label":"Wang et al.","section":"sources","anchor":"ref-wang2006","href":"https://doi.org/10.1016/j.cell.2006.11.023"}]},{"id":"rna-polymerase-ppi-power-stroke-debate","machine":"rna-polymerase","kind":"qa","prompt":"Which model of RNA polymerase movement did optical-trap force-velocity data favour over a power stroke tied to pyrophosphate release?","answer":"A Brownian ratchet with a second NTP site.","explanation":"The power stroke fitted poorly (reduced χ² 6.03), measured on E. coli RNA polymerase. The machine notes still say pyrophosphate release drives the chain forward.","section":"evidence","topic":"debate","sources":["evidence:brownian-ratchet-force-velocity","step:Pyrophosphate leaves","machine:energy","ref:abbondanzieri2005"],"tags":["ratchet","pyrophosphate"],"difficulty":3,"url":"/machines/rna-polymerase#evidence","cites":[{"source":"evidence:brownian-ratchet-force-velocity","machine":"rna-polymerase","label":"Mechanism that fits the force-velocity data (Abbondanzieri EA 2005)","section":"evidence","anchor":"ev-brownian-ratchet-force-velocity"},{"source":"step:Pyrophosphate leaves","machine":"rna-polymerase","label":"Step: Pyrophosphate leaves","section":"mechanism"},{"source":"machine:energy","machine":"rna-polymerase","label":"Summary","section":"summary"},{"source":"ref:abbondanzieri2005","machine":"rna-polymerase","label":"Abbondanzieri et al.","section":"sources","href":"https://doi.org/10.1038/nature04268"}]}]}