Cloze Drill
50 fill-in-the-blank cards Β· Ch. 6–12 Β· type the missing term and hit check
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transcriptionRNA Polymerase & Initiation
Bacteria haveRNA polymerase; eukaryotes have up to(including organellar).
In bacteria, thefactor recognizes theandpromoter elements.
RNAP I transcribes, RNAP II transcribes, and RNAP III transcribesand small RNAs.
For RNAP II initiation, TBP (part of) binds the~25 bp upstream of the start site.
The CTD of RNAP II isduring initiation, andby TFIIH during elongation, recruiting mRNA processing enzymes.
transcriptionTermination
Rho-independent termination requires a GC-richfollowed by a run ofnucleotides. Both are required.
In Rho-dependent termination, theprotein binds the emerging mRNA and chases RNAP. When RNAP pauses, Rho causes RNA to be.
transcriptionSpecific TFs & Epigenetics
Specific TFs bindor repressor sequences β€” near OR up toaway from the promoter.
Activator TFs can recruit(histone acetyltransferases) to open chromatin, or cause DNA to loop so they contact thecomplex at the promoter.
Acetylation of lysines on H3/H4 tails =chromatin. H3K9=chromatin.
⚠ TRAP: H3K9 acetyl = ACTIVE but H3K9 methyl = INACTIVE β€” same lysine, opposite outcome.
DNA methylation adds CH₃ to theposition of cytosine atdinucleotides. Used in X-inactivation,, and repressing transposons.
The 4 types of DNA-binding domains in TFs: zinc finger,,, and helix-loop-helix.
A loss-of-function mutation causing INCREASED expression = protein was a. Mutation causing DECREASED expression = protein was a.
processingmRNA Processing β€” Order & Mechanism
The three mRNA processing events in order: (1), (2), (3). All occur in the.
The 5β€² cap is a(m7G) added via an unusualtriphosphate linkage.
⚠ TRAP: 5β€²-to-5β€² linkage (unusual!) β€” not the normal 5β€²-to-3β€².
Poly-A tail: RNA cleaved at thesignal, then Poly(A) Polymerase () adds ~80–250 A's using.
The spliceosome consists ofsnRNPs (U1, U2, U4, U5, U6). U1 binds the(GU). U2 binds theA, forming a. Intron ends withat 3β€² SS.
The lariat forms via aphosphodiester linkage between the 5β€² end of the intron and the 2β€²OH of the branch point.
⚠ TRAP: 2β€²-5β€² linkage β€” not the normal 3β€²-5β€². The branch point is always A.
translationRibosomes & Initiation
Prokaryotic ribosomes =(30S + 50S). Eukaryotic ribosomes =(40S + 60S). Biggest functional difference is in.
Prokaryotes find the start codon via thesequence, base-pairing with. Eukaryotes useto find the first AUG.
Prokaryotes use(N-formylmethionine) as the first amino acid. Eukaryotes use regular. Prokaryotes useIFs; eukaryotes use at leasteIFs.
⚠ TRAP: fMet is ONLY in prokaryotes (and mito/chloro initiation).
Three tRNA sites in the ribosome: A (β€” incoming), P (β€” growing chain), E (β€” leaving).
translationElongation & Termination
Step 1 β€” Decoding: EF-Tu (prok) /(euk) delivers charged tRNA + GTP to the A-site. GTP hydrolysis = proofreading gate.
Step 2 β€” Transpeptidation: Peptide bond formed by, which is actually theitself β€” a ribozyme, NOT a protein enzyme.
⚠ TRAP: Peptidyl transferase = ribosomal RNA (ribozyme). Don't say it's a protein.
Step 3 β€” Translocation: EF-G (prok) /(euk) + GTP moves ribosome 3 nt forward. Each elongation cycle costsGTPs total.
When a stop codon enters the A-site, aprotein enters β€” triggering polypeptide release and dissociation of ribosomal.
⚠ TRAP: Release factors are PROTEINS, not tRNAs.
translationProtein Modifications
N-linked glycosylation: 14-sugar unit added to theofviain the ER.
O-linked glycosylation: single sugars added to theof. Occurs in the.
Phosphorylation adds phosphate fromto Ser, Thr, orvia a kinase. Removed by a. Reversible switch.
Myristate β†’ N-terminal. Palmitate β†’ sulfur of. Prenyl group β†’ Cys near. GPI anchor β†’ C-terminus in.
sortingSecretory Pathway & ER Targeting
Secreted protein pathway: ribosome starts β†’ N-terminalemerges β†’binds, halts translation β†’ docks at ER β†’threads protein into lumen β†’ signal cleaved.
Secretory route: ER β†’β†’β†’ medial β†’ trans Golgi β†’(TGN) β†’ vesicle β†’ plasma membrane β†’.
⚠ TRAP: Don't skip ERGIC β€” Rowen tests this step explicitly.
Lysosomal proteins are tagged in the Golgi with(M6P) β†’ packaged into-coated vesicles β†’ endosomes β†’ lysosomes. Without M6P they areinstead.
sortingVesicle Types & Fusion
COPI vesicles transport(retrograde). COPII transport(anterograde). Clathrin fromto rest of cell AND plasma membrane β†’ endosome.
Vesicle formation: G protein+ GTP recruits adaptor β†’ adaptor binds cargo receptor β†’ recruits coat proteins β†’ membrane buds off.
Vesicle fusion: Rab protein bindson target membrane. Then(vesicle) intertwines with(target) β†’ membranes fuse.
sortingNuclear, Mito, Chloroplast, Peroxisome Import
Nuclear import: importin bindson the protein β†’ transported through NPC β†’ in nucleus,binds importin β†’ protein.
⚠ TRAP: NLS can be ANYWHERE on the protein. mRNA export does NOT use the Ran system.
Nucleus, mito, chloroplast, and peroxisome proteins are made on,imported, with NOused.
Mito import signal =(N-terminal). Chloroplast signal =. Peroxisome matrix signal =(C-terminal SKL) or PTS2 (N-terminal).
Mitochondria: 2 membranes, compartments = IMS +. Chloroplast: 3 membranes (outer + inner +), 3 compartments (IMS ++ thylakoid lumen).
⚠ rowen trapsHigh-frequency exam traps
CTD of RNAP II isduring initiation andduring elongation. Phosphorylation by.
Peptidyl transferase is theβ€” a ribozyme. It is NOT aenzyme.
When a stop codon is reached, a(release factor) enters the A-site β€” NOT a.
The NLS can be locatedon the protein β€” NOT restricted to the.
mRNA export from the nucleus doesuse thesystem. Ran-GTP gradient is used forimport/export only.
H3K9 acetylation =chromatin. H3K9 methylation =chromatin. Same lysine, different modification = opposite outcome.
Without, lysosomal proteins will beinstead of going to lysosomes.
The 5β€² cap is attached via atriphosphate linkage β€” NOT 5β€²-to-3β€². Rho-independent termination requires BOTH theAND a run ofresidues β€” neither alone is sufficient.
COPI =(retrograde). COPII =(anterograde). Clathrin = TGN outward ANDfrom plasma membrane.
In prokaryotes, transcription and translation arebecause there is noseparating them. This cannot happen in eukaryotes.
Short Answer Quiz
8 Rowen-style mechanism questions β€” write your answer, then reveal the rubric and self-score.
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Ch 6 3 pts SA–1
Describe the mechanism of pre-mRNA splicing by the spliceosome. Include: (1) the two transesterification reactions, (2) the branch point A and the lariat intermediate, and (3) which snRNPs are responsible for recognizing the 5β€² splice site vs. the branch point.
RUBRIC β€” KEYWORDS REQUIRED
  • Rxn 1: 2β€²-OH of branch-point A attacks 5β€² splice site β†’ lariat intermediate + free 3β€²-OH on exon 1
  • Rxn 2: Free 3β€²-OH of exon 1 attacks 3β€² splice site β†’ exons joined, lariat released
  • U1 snRNP recognizes 5β€² splice site; U2 snRNP recognizes branch point (via base-pairing)
  • U4/U6 unwind to activate catalytic center; U5 holds exon ends
  • No ATP consumed in transesterification steps (bonds exchanged, not broken and formed)
My score:
Ch 6 3 pts SA–2
The cleavage/polyadenylation machinery adds a poly-A tail to pre-mRNA. Explain: (1) what sequence is recognized, (2) what cuts the RNA, and (3) why the poly-A tail protects mRNA from degradation.
RUBRIC β€” KEYWORDS REQUIRED
  • AAUAAA is the poly-A signal sequence recognized by CPSF (cleavage and polyadenylation specificity factor)
  • CstF binds downstream GU-rich element; endonucleolytic cleavage occurs 10–30 nt downstream of AAUAAA
  • Poly-A polymerase (PAP) adds ~250 A residues (template-independent)
  • Poly-A tail bound by PABP (poly-A binding protein) β†’ protects 3β€² end from exonucleases + stimulates translation via interaction with eIF4G
  • NOT "the tail is a cap" β€” cap is at 5β€² end and is structurally different
My score:
Ch 7 3 pts SA–3
Explain how histone acetylation leads to transcriptional activation. Your answer must include: (1) the enzyme that adds acetyl groups and where it acts, (2) the physical reason acetylation opens chromatin, and (3) how bromodomain proteins connect acetylation to the transcription machinery.
RUBRIC β€” KEYWORDS REQUIRED
  • HATs (histone acetyltransferases, e.g., p300/CBP) add acetyl group to lysine residues on histone tails (especially H3K9ac, H3K27ac)
  • Neutralizes positive charge on lysine β†’ reduces electrostatic attraction between histone and negatively charged DNA β†’ nucleosome loosens
  • Bromodomain proteins (e.g., TAF1 in TFIID) recognize acetyl-lysine β†’ recruit general transcription factors or Mediator β†’ forms PIC at promoter
  • HDACs (deacetylases) reverse this β†’ repression
  • Acetylation is NOT permanent; it is a reversible epigenetic mark
My score:
Ch 8 3 pts SA–4
Describe cotranslational import into the ER. Start from the signal peptide emerging from the ribosome and end with the signal peptide being cleaved. Name every molecular player in the correct order.
RUBRIC β€” ORDER MATTERS
  • Signal peptide (N-terminal hydrophobic sequence) emerges from ribosome β†’ recognized by SRP (signal recognition particle)
  • SRP binds signal peptide β†’ translation paused (elongation arrest)
  • SRP-ribosome complex docks at SRP receptor on ER membrane β†’ GTP hydrolysis releases SRP
  • Ribosome transferred to translocon (Sec61 complex) β†’ translation resumes
  • Polypeptide threads through aqueous translocon channel into ER lumen
  • Signal peptidase (ER luminal enzyme) cleaves signal peptide co-translationally
  • Mature protein released into ER lumen for folding/glycosylation
My score:
Ch 8 3 pts SA–5
Compare ERAD (ER-associated degradation) and autophagy as quality control mechanisms. For each: what triggers it, what machinery executes it, and what is the final degradative compartment?
RUBRIC β€” COMPARISON FORMAT
  • ERAD: Triggered by misfolded protein in ER (detected by BiP/GRP78 chaperone + calnexin). Retrotranslocated through Sec61/Hrd1 into cytosol β†’ polyubiquitinated by E3 ligase (HRD1) β†’ degraded by 26S proteasome
  • Autophagy: Triggered by nutrient stress, damaged organelles, or protein aggregates too large for proteasome. Double-membrane autophagosome (nucleated by ULK1 complex + PI3K) engulfs cargo β†’ fuses with lysosome β†’ hydrolases degrade contents
  • Key difference: ERAD = single misfolded proteins β†’ proteasome. Autophagy = bulk/large cargo β†’ lysosome
  • Both involve ubiquitin tagging for selective recognition (p62/SQSTM1 adapter in selective autophagy)
My score:
Ch 11 3 pts SA–6
Explain the COPII vesicle cycle from ER to Golgi. Include: what cargo is selected, what GTPase drives budding, how the coat is shed, and how the vesicle is targeted to the correct membrane.
RUBRIC β€” MECHANISM CHAIN
  • Sar1-GTP (ARF-like GTPase) inserts into ER membrane β†’ recruits Sec23/24 (inner coat, cargo selection) β†’ then Sec13/31 (outer cage)
  • Sec24 directly binds ER export signals on cargo proteins
  • Sar1 GAP activity (by Sec23) hydrolyzes GTP β†’ coat disassembles after budding
  • v-SNAREs (e.g., Sec22) on vesicle pair with t-SNAREs (e.g., Sed5) on cis-Golgi β†’ SNARE zippering drives membrane fusion
  • Tethering factors (e.g., p115, GM130) capture the vesicle at the Golgi before SNARE pairing
  • COPI (retrograde) is different from COPII (anterograde ERβ†’Golgi)
My score:
Ch 12 3 pts SA–7
Trace the path of electrons through the mitochondrial ETC from NADH to Oβ‚‚. For each complex (I, II, III, IV): state what enters, what leaves, and whether protons are pumped across the inner mitochondrial membrane.
RUBRIC β€” EACH COMPLEX MUST BE ADDRESSED
  • Complex I (NADH dehydrogenase): NADH β†’ NAD⁺; electrons β†’ ubiquinone (Q) β†’ QHβ‚‚; pumps 4H⁺ to IMS
  • Complex II (succinate dehydrogenase): FADHβ‚‚ β†’ FAD; electrons β†’ QHβ‚‚; NO proton pumping (embedded in IMM but no proton channel)
  • Complex III (cytochrome bc1): QHβ‚‚ β†’ cytochrome c; pumps 4H⁺ via Q cycle; electrons pass one at a time
  • Complex IV (cytochrome c oxidase): Cytochrome c β†’ Oβ‚‚ (final acceptor); Oβ‚‚ + 4e⁻ + 4H⁺ β†’ 2Hβ‚‚O; pumps 2H⁺
  • Total proton gradient drives ATP synthase (Complex V); ~2.5 ATP/NADH, ~1.5 ATP/FADHβ‚‚
My score:
Ch 6–12 3 pts SA–8 ⚑ Integration
A researcher treats cells with a drug that blocks all nuclear export via the NPC. Predict the downstream consequences for: (1) mRNA translation, (2) ribosome biogenesis, and (3) mitochondrial function. Explain the mechanistic link for each.
RUBRIC β€” INTEGRATION (MECHANISM REQUIRED FOR EACH)
  • mRNA translation: mRNAs cannot exit nucleus β†’ ribosomes in cytoplasm have no new mRNA templates β†’ protein synthesis halts (only existing mRNAs/proteins persist until degraded)
  • Ribosome biogenesis: Pre-rRNA processed in nucleolus β†’ assembled into pre-40S and pre-60S subunits β†’ exported via NPC using Ran-GTP–dependent exportins. Export blocked β†’ no new cytoplasmic ribosomes β†’ translation further reduced in a compounding effect
  • Mitochondrial function: ~99% of mitochondrial proteins are nuclear-encoded β†’ translated in cytoplasm β†’ imported post-translationally. No new nuclear-encoded ETC subunits/assembly factors β†’ existing ETC complexes degrade without replacement β†’ oxidative phosphorylation fails over time
  • Bonus: Only 13 ETC proteins are mitochondria-encoded (remain functional briefly) β€” this is the Rowen trap
My score:
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