Overview Diagrams
Central Dogma: DNA → RNA → Protein → Sorting → Destination
Ch6: RNA Synthesis and Processing
Vocabulary (26 terms)
Enzyme → reads DNA template 3'→5', synthesizes RNA 5'→3' using NTPs (ATP, UTP, GTP, CTP) as substrates. No primer needed. Prok: 1 RNA pol (5 subunits: α₂ββ'ω). Euk: Pol I, II, III.
Key facts: Transcription bubble = ~12-14 bp unwound DNA. Error rate ~10⁻⁴. β subunit = catalytic site (phosphodiester bond formation), β' = DNA binding, α = assembly + UP element contacts, ω = assembly/stability
Prok initiation subunit → binds core enzyme (α₂ββ'ω) to form holoenzyme. σ⁷⁰ = primary/housekeeping factor. Region 4.2 contacts -35 (TTGACA); region 2.4 contacts -10 (TATAAT). Melts ~12 bp at -10 to form open complex.
Key facts: σ70 recognizes -10 (TATAAT) and -35 (TTGACA) elements; dissociates after ~10 nt synthesized
Bacterial RNA polymerase minus the σ subunit (α₂ββ'ω); can elongate but not initiate specifically
Key facts: α₂ββ'ω composition: β' binds DNA, β holds active site, α assembles enzyme + contacts UP element
Cis-regulatory DNA element at/near +1 start site where RNA pol + factors assemble. Prok: -10 (TATAAT), -35 (TTGACA), UP element (A/T rich, contacts α-CTD). Euk Pol II: TATA box (-25 to -30), Inr (overlaps +1), BRE, DPE, MTE.
Key facts: Promoter strength = frequency of transcription initiation. Strong promoter = consensus -10/-35 match. Only 10-20% of Pol II promoters have TATA box; CpG-island promoters lack TATA, use Inr/DPE instead
TATAAT consensus sequence ~10 bp upstream of transcription start in bacteria
Key facts: AT-rich → low melting temp → easy strand separation for open complex formation at +1
TTGACA consensus sequence ~35 bp upstream of transcription start; recognized by σ
Key facts: σ region 4.2 contacts -35; spacing between -35 and -10 = 17±1 bp (critical for alignment)
TATAA sequence ~25-30 bp upstream of eukaryotic Pol II transcription start; bound by TBP
Key facts: Bound by TBP (saddle shape, minor groove); bends DNA ~80°; positions +1 start site
Proteins (TFIID, TFIIB, TFIIF, TFIIE, TFIIH) required for Pol II to initiate transcription
Key facts: Ordered assembly: TFIID → TFIIB → TFIIF+PolII → TFIIE → TFIIH; forms preinitiation complex (PIC)
TFIID contains TBP (TATA-binding protein) + TAFs; first factor to bind the promoter
Key facts: TBP binds TATA box; TAFs (TBP-associated factors) recognize Inr, DPE, MTE core promoter elements
Dual-function GTF: (1) XPB subunit = 3'→5' helicase, unwinds ~12-14 bp at +1 for open complex. (2) Cdk7/cyclin H = kinase, phosphorylates CTD Ser5 → promoter escape + capping enzyme recruitment. Also functions in NER DNA repair (XPB/XPD mutations → xeroderma pigmentosum).
Key facts: 10 subunits total. XPB mutation → trichothiodystrophy (TTD) or Cockayne syndrome. α-amanitin does NOT inhibit TFIIH; it inhibits Pol II directly
Repeated heptad (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) on Pol II largest subunit; 52 repeats in humans
Key facts: 52 heptad repeats (humans); Ser5-P by TFIIH → capping enzyme recruitment; Ser2-P by P-TEFb (Cdk9/cyclin T) → elongation/splicing/poly-A factors
Large multi-subunit complex bridging gene-specific transcription factors and Pol II
Key facts: ~30 subunits; head module contacts Pol II CTD, tail module contacts gene-specific activators; required for regulated transcription
Transcribes 45S pre-rRNA (yields 28S, 18S, 5.8S rRNAs) in the nucleolus
Key facts: Transcribes ~400 tandem rDNA repeats in nucleolus; produces >80% of total cellular RNA by mass
Transcribes tRNA, 5S rRNA, and some snRNAs
Key facts: Internal promoter (type 1/2) for tRNA/5S rRNA; upstream promoter (type 3) for U6 snRNA
Modified guanosine added to the 5' end of pre-mRNA co-transcriptionally
Key facts: Added co-transcriptionally after ~20-30 nt; 5'-5' triphosphate linkage; bound by CBC (nuclear) then eIF4E (translation)
~200 adenines added to 3' end of mRNA by poly-A polymerase (PAP) — template-independent. Functions: (1) stabilizes mRNA vs 3'→5' exonucleases, (2) aids nuclear export, (3) enhances translation (PABP-eIF4G interaction)
Key facts: CPSF binds AAUAAA → CstF binds GU-rich element → cleavage → PAP adds ~200 A's; PABP coats tail → stabilizes + aids export
Non-coding intervening sequence removed from pre-mRNA by splicing
Key facts: Average human gene: ~8 introns; some span >100 kb; begin GU, end AG (GT-AG rule in DNA); removed by spliceosome
Coding sequence retained in mature mRNA after splicing
Key facts: Joined after intron removal; can be coding, 5' UTR, or 3' UTR; exon shuffling drives protein domain evolution
5 snRNPs (U1, U2, U4, U5, U6) + ~200 proteins = megadalton complex. Catalytic core = U2+U6 snRNAs (ribozyme, no protein catalysis). Two transesterification reactions: (1) 2'-OH of branch-point A attacks 5' splice site → lariat, (2) free 3'-OH of exon 1 attacks 3' splice site → exons ligated, lariat released + debranched + degraded
Key facts: 5 snRNPs + ~200 proteins; catalytic core = U2/U6 snRNAs (ribozyme); two transesterifications → lariat + ligated exons
Small nuclear ribonucleoprotein particles (U1-U6) that form the spliceosome
Key facts: U1 base-pairs with 5' splice site; U2 base-pairs with branch point (bulges A); U6 replaces U1 and catalyzes reaction
Self-splicing intron using an external guanosine as nucleophile; found in rRNA of Tetrahymena
Key facts: External G cofactor attacks 5' splice site; 3'-OH of upstream exon attacks 3' splice site; found in Tetrahymena rRNA (Cech, 1982)
Self-splicing intron using an internal adenosine 2'-OH; produces a lariat
Key facts: Internal branch-point A (2'-OH) attacks 5' splice site → lariat; same chemistry as spliceosome; supports RNA world hypothesis
One gene → multiple mRNA variants by differential exon inclusion/exclusion. Types: exon skipping (most common), alt 5'/3' splice site, intron retention, mutually exclusive exons. >95% of human multi-exon genes undergo alt splicing
Key facts: SR proteins (Ser/Arg-rich) bind ESEs (exonic splicing enhancers) → promote exon inclusion. hnRNPs bind ESSs (exonic splicing silencers) → promote exon exclusion/skipping. Classic example: Drosophila Dsx (sex determination)
Small nucleolar ribonucleoprotein particles that guide rRNA modification (methylation, pseudouridine)
Key facts: Box C/D snoRNPs guide 2'-O-methylation; Box H/ACA snoRNPs guide pseudouridylation; ~200 modifications per rRNA
Ribozyme that cleaves the 5' leader of pre-tRNA
Key facts: RNA subunit = catalytic component; cleaves 5' leader of pre-tRNA; shared Nobel Prize with Cech (1989)
AAUAAA sequence in pre-mRNA that signals cleavage and poly-A addition ~20 nt downstream
Key facts: CPSF recognizes AAUAAA; CstF binds downstream GU-rich element; cleavage occurs ~10-30 nt after AAUAAA
Mechanisms (5)
Transcription by RNA Polymerase II
mRNA Processing Pipeline (5' cap → splicing → 3' poly-A)
Spliceosome Assembly and Catalysis
rRNA Processing in the Nucleolus
Self-Splicing Introns: Group I vs Group II
Comparison Tables (4)
RNA Polymerase I vs II vs III
| Feature | RNA Pol I | RNA Pol II | RNA Pol III |
|---|---|---|---|
| Products | 28S, 18S, 5.8S rRNA | mRNA, miRNA, lncRNA, most snRNA | tRNA, 5S rRNA, some snRNA |
| Location | Nucleolus | Nucleoplasm | Nucleoplasm |
| Promoter type | Upstream (UBF + SL1) | TATA box, Inr, BRE, DPE | Internal (5S, tRNA) or upstream (U6) |
| α-amanitin sensitivity | Insensitive | Very sensitive | Moderately sensitive |
| CTD present? | No | Yes (52 heptad repeats in humans) | No |
| Products capped? | No | Yes (7-methylguanosine) | No |
| Products polyadenylated? | No | Yes | No |
Pre-mRNA vs Mature mRNA
| Feature | Pre-mRNA | Mature mRNA |
|---|---|---|
| Location | Nucleus | Cytoplasm |
| 5' end | Triphosphate → capped during transcription | 7-methylguanosine cap |
| 3' end | Extends past poly-A signal | Cleaved + poly-A tail (~200 A's) |
| Introns | Present | Removed by splicing |
| Size | Larger (includes introns + UTRs) | Smaller (exons + UTRs only) |
| Associated proteins | hnRNPs, splicing factors | mRNPs, eIF4E, PABP |
Group I vs Group II Self-Splicing Introns
| Feature | Group I | Group II |
|---|---|---|
| Nucleophile | External free guanosine (G-OH) | Internal adenosine 2'-OH |
| Intermediate | Linear | Lariat |
| Product released | Linear intron | Lariat intron |
| Found in | Tetrahymena rRNA, some organelle genes | Organelle genes, some bacteria |
| Resembles spliceosome? | No | Yes — evolutionary ancestor model |
| Cofactor needed | Free G nucleoside | None (internal A) |
Prokaryotic vs Eukaryotic Transcription
| Feature | Prokaryotic | Eukaryotic |
|---|---|---|
| RNA polymerases | One (core + σ) | Three (Pol I, II, III) |
| Initiation factor | σ factor | GTFs (TFIID, B, F, E, H) |
| Promoter elements | -10, -35 | TATA box, Inr, BRE, DPE + enhancers |
| mRNA processing | None (translated co-transcriptionally) | Capping, splicing, polyadenylation |
| Coupling | Transcription + translation coupled | Transcription and translation separated by nuclear envelope |
| Introns | Rare | Common (avg ~8 per gene) |
| Termination | Rho-dependent or intrinsic (stem-loop) | Poly-A signal + torpedo/allosteric model |
Diagrams (6)
Transcription Bubble
mRNA Processing Pipeline
Spliceosome Assembly
RNA Polymerase Comparison
Self-Splicing Introns
CTD Phosphorylation Code: Ser5 vs Ser2
Exam Traps (10)
Ch7: Transcriptional Regulation and Epigenetics
Vocabulary (25 terms)
Prok gene cluster: promoter-operator-structural genes, transcribed as single polycistronic mRNA. Lac operon = lacZ (β-galactosidase), lacY (permease), lacA (transacetylase). Dual control: negative (LacI repressor) + positive (CAP-cAMP)
Key facts: Allows coordinate regulation of metabolic pathways
Tetramer encoded by lacI gene (constitutively expressed). Binds operator (21-bp palindrome overlapping +1) → physically blocks RNA pol. Allolactose (lactose isomer) = inducer → binds LacI → allosteric conformational change → LacI releases operator → transcription proceeds
Key facts: Classic model of negative regulation; allolactose binding releases it
Cis-regulatory DNA element overlapping/adjacent to promoter. Lac: 21-bp palindrome at +1, repressor binds via helix-turn-helix motif. trp operon: operator + attenuator (leader peptide senses Trp-tRNA levels)
Key facts: The molecular OFF switch in negative control
Positive regulator (homodimer, helix-turn-helix). Low glucose → adenylyl cyclase active → cAMP rises → cAMP binds CAP → CAP-cAMP binds DNA upstream of promoter (-61) → bends DNA ~90° → enhances RNA pol α-CTD contact → 50-fold activation
Key facts: Classic model of positive control; links carbon source availability to gene expression
Cis-regulatory element containing clustered TF binding sites. Works at any distance (up to 1 Mb), any orientation (upstream/downstream/within introns). Contacts promoter via DNA looping (cohesin + CTCF define loop boundaries). Active enhancers marked by H3K27ac + H3K4me1
Key facts: Contains binding sites for multiple TFs; can work in any orientation and distance
Sequence-specific TF with modular structure: DNA-binding domain (DBD) + activation domain (AD). DBD types: helix-turn-helix, zinc finger (Cys₂His₂, 1 Zn²⁺ per finger), leucine zipper (coiled-coil dimerization), helix-loop-helix (HLH, homo/heterodimers). AD recruits Mediator/HATs/remodelers
Key facts: DBD types: helix-turn-helix, zinc finger (Cys₂His₂, 1 Zn²⁺ per finger), leucine zipper (coiled-coil dimerization + basic region), HLH (helix-loop-helix, homo/heterodimers). Methods: EMSA/gel shift (detects TF-DNA binding in vitro), DNase footprinting (identifies exact binding site on DNA), ChIP-seq (maps TF binding genome-wide in vivo), reporter gene assays (luciferase/GFP downstream of promoter/enhancer)
Negative TF that silences genes. Mechanisms: (1) recruit HDACs → deacetylate histones → compact chromatin, (2) recruit HMTs (e.g., SUV39H1 → H3K9me3, EZH2/PRC2 → H3K27me3), (3) compete with activator for same DNA site, (4) mask activator's AD (quenching). Example: Rb recruits HDAC to E2F-target promoters
Key facts: Polycomb group (PcG) = major eukaryotic repressors. PRC2 (EZH2 catalytic subunit) = writer (trimethylates H3K27 → H3K27me3). PRC1 = reader (chromodomain binds H3K27me3) + E3 ligase (ubiquitylates H2AK119). Trithorax group (TrxG) = antagonizes Polycomb, maintains active state. Rb recruits HDAC to E2F-target promoters → blocks cell cycle entry
~30-subunit complex with 4 modules: head (contacts Pol II CTD), middle (scaffold), tail (contacts gene-specific activators/enhancers), kinase module (CDK8, dissociable, can repress). Essential for converting activator signals into Pol II recruitment
Key facts: Required for ~90% of Pol II genes. CDK8 kinase module (dissociable 4th module) can phosphorylate TFs → positive or negative regulation. Head module contacts Pol II CTD, tail contacts activators. Mediator mutations → developmental disorders (MED12 mutations → FG/Opitz-Kaveggia syndrome, intellectual disability)
Physical bending of DNA that allows distant enhancer-bound TFs to contact promoter-bound factors
Key facts: Explains how enhancers 1000 kb away regulate their target genes
Ring-shaped protein complex that stabilizes DNA loops between enhancers and promoters
Key facts: SMC1/SMC3 heterodimer + RAD21 (kleisin) + SA subunit form ring that topologically embraces DNA. Loop extrusion: cohesin slides along DNA creating loops until blocked by convergent CTCF sites. Cohesin mutations → Cornelia de Lange syndrome (cohesinopathy). Also mediates enhancer-promoter contacts
Architectural protein that defines boundaries of chromosomal loop domains (with cohesin)
Key facts: 11 zinc fingers → recognizes ~20 bp consensus motif. Binds DNA in orientation-dependent manner (convergent CTCF sites block cohesin). Insulator function: prevents enhancer from activating wrong gene across TAD boundary. CTCF binding is methylation-sensitive (CpG methylation blocks CTCF → boundary lost → ectopic enhancer activation → disease, e.g., IDH-mutant gliomas)
HATs (p300/CBP, GCN5, PCAF) transfer acetyl group from acetyl-CoA to ε-amino group of lysine on H3/H4 tails. Key residues: H3K9ac, H3K14ac, H3K27ac (enhancer mark), H4K16ac. Neutralizes Lys + charge → weakens histone-DNA electrostatic grip → open chromatin (euchromatin)
Key facts: Bromodomain proteins (BRD4) = acetyl-lysine readers → recruit P-TEFb for transcription elongation. p300/CBP mutations → Rubinstein-Taybi syndrome. HDAC inhibitors (vorinostat, romidepsin) = FDA-approved cancer drugs (cause hyperacetylation → reactivate tumor suppressors). Acetyl-CoA = acetyl donor
Removal of acetyl groups from histones by histone deacetylases
Key facts: Restores positive charge → tightens chromatin → gene SILENCING
ATP-dependent machines (e.g., SWI/SNF) that slide, eject, or restructure nucleosomes
Key facts: Required to expose promoters/enhancers buried in nucleosomes
Decondensed, DNase I-sensitive chromatin. Marks: H3K4me3 (active promoter), H3K27ac (active enhancer), H3K36me3 (active gene body), hyperacetylated H3/H4. Replicates early in S phase. Contains ~92% of genes
Key facts: 10-nm fiber (beads-on-a-string) configuration. DNase I hypersensitive sites mark regulatory elements. Replicates early S phase. Accessible to TFs, RNA pol, remodelers. Contains ~92% of protein-coding genes
Condensed, DNase I-resistant, transcriptionally silent. 2 types: (1) Constitutive = permanent (centromeres, telomeres, satellite DNA), marked by H3K9me3 → HP1 binding. (2) Facultative = reversible (inactive X, imprinted genes), marked by H3K27me3 → Polycomb repressive complex (PRC2)
Key facts: Constitutive: centromeres (α-satellite DNA), telomeres, pericentromeric regions — marked by H3K9me3, bound by HP1 (chromodomain). Facultative: inactive X (Barr body, coated by Xist lncRNA), imprinted loci, Polycomb-silenced genes — marked by H3K27me3. Replicates late S phase. Position effect variegation: gene near heterochromatin boundary → stochastic silencing
DNMT3a/DNMT3b = de novo methyltransferases (establish new patterns). DNMT1 = maintenance methyltransferase (copies pattern to daughter strand after replication, recognizes hemimethylated CpG via UHRF1). Methyl group added to C5 of cytosine → 5-methylcytosine (5mC)
Key facts: Promoter CpG methylation → recruits MeCP2 + NCoR/HDAC complex → chromatin compaction → silencing. Gene body methylation correlates with active transcription. TET enzymes (TET1/2/3) = erasers: oxidize 5mC → 5hmC → 5fC → 5caC → base excision repair → unmodified C. 5-azacytidine/decitabine = DNMT inhibitors (cancer drugs, reactivate silenced tumor suppressors)
Region of DNA with high frequency of CG dinucleotides; often found at gene promoters
Key facts: When unmethylated → gene active; when methylated → gene silenced
Heritable changes in gene expression NOT due to changes in DNA sequence (e.g., methylation, histone marks)
Key facts: Explains how identical DNA in all cells produces different cell types
Silencing of one X chromosome in female mammals via Xist lncRNA and heterochromatin formation
Key facts: Xist lncRNA (~17 kb) coats inactive X in cis → recruits PRC2 (H3K27me3) + DNMT3b (DNA methylation) → heterochromatin (Barr body). Tsix = antisense lncRNA, blocks Xist on active X. Random in somatic cells (chosen ~day 5.5 in mouse). ~15% of genes escape inactivation. XCI = dosage compensation in XX mammals
Parent-of-origin gene expression: one allele silenced by DNA methylation established in germline. ~100 imprinted genes in humans. IGF2 = paternally expressed (maternal allele silenced). H19 = maternally expressed (paternal allele silenced)
Key facts: ~100 imprinted genes in humans. Prader-Willi = loss of paternal 15q11-13 (obesity, intellectual disability). Angelman = loss of maternal UBE3A at same locus (seizures, ataxia). Beckwith-Wiedemann = IGF2 overexpression (overgrowth). Imprint marks erased in primordial germ cells, re-established based on sex of new parent during gametogenesis. ICRs (imprinting control regions) regulate via CTCF-dependent insulators
~22 nt non-coding RNA that binds 3' UTR of target mRNA → translational repression + mRNA degradation
Key facts: Seed region (nt 2-8 of guide strand) base-pairs with 3' UTR of target mRNA (imperfect match = miRNA; perfect match = siRNA-like cleavage by Ago2). 1 miRNA can target ~hundreds of mRNAs. Mechanism: blocks ribosome scanning + recruits deadenylase (CCR4-NOT) → mRNA decapping → degradation in P-bodies. ~60% of human genes are miRNA targets. Fire & Mello 2006 Nobel (RNAi in C. elegans)
~22 nt double-stranded RNA that targets perfectly complementary mRNA for cleavage by RISC/Argonaute
Key facts: Used experimentally for gene knockdown (RNAi); also endogenous defense
Effector complex for RNAi. Core = Argonaute (Ago2 in humans has Slicer/endonuclease activity). Guide strand (antisense) loaded; passenger strand degraded. miRNA-RISC: imperfect match → translational repression. siRNA-RISC: perfect match → Ago2 cleaves mRNA between nt 10-11 of guide
Key facts: Ago2 = only human Argonaute with Slicer (endonuclease) activity. Slicer cleaves target mRNA between nt 10-11 of guide strand (requires perfect complementarity). RISC is catalytic — each complex degrades multiple mRNA molecules. siRNA-RISC: perfect match → cleavage. miRNA-RISC: imperfect match → translational repression + deadenylation
Combinatorial modifications on histone tails (acetylation, methylation, phosphorylation, ubiquitylation) form a code read by effector proteins. Writers: HATs, HMTs, kinases. Erasers: HDACs, HDMs (e.g., LSD1, JMJD), phosphatases. Readers: bromodomains (acetyl), chromodomains (methyl), PHD fingers
Key facts: Active marks: H3K4me3 (promoter), H3K36me3 (gene body), H3K27ac (enhancer). Silencing marks: H3K9me3 (HP1 → constitutive heterochromatin), H3K27me3 (Polycomb → facultative). Bivalent domains = H3K4me3 + H3K27me3 at same promoter in stem cells (poised genes). Writers: HATs, HMTs (SET domain). Readers: bromodomains (Ac-Lys), chromodomains (Me-Lys), PHD fingers. Erasers: HDACs, KDMs (LSD1, JmjC family)
Mechanisms (5)
Transcriptional Activation via Enhancer → Mediator → Pol II
Chromatin Remodeling: HATs Open / HDACs Close
DNA Methylation → Gene Silencing
miRNA Pathway (Gene Silencing)
Epigenetic Inheritance Through Cell Division
Comparison Tables (5)
Activators vs Repressors
| Feature | Activators | Repressors |
|---|---|---|
| Effect | Stimulate transcription | Inhibit transcription |
| Mechanism | Recruit Mediator, HATs, Pol II | Recruit HDACs, block activator binding |
| Chromatin effect | Open (euchromatin) | Close (heterochromatin) |
| Histone marks recruited | Acetylation (H3K27ac, H3K9ac) | Methylation (H3K9me3, H3K27me3) |
| Example | CAP in E. coli; p53 in humans | lac repressor; Rb in humans |
HATs vs HDACs
| Feature | HATs | HDACs |
|---|---|---|
| Full name | Histone acetyltransferases | Histone deacetylases |
| Reaction | Add acetyl groups to lysines | Remove acetyl groups from lysines |
| Effect on charge | Neutralize positive charge | Restore positive charge |
| Chromatin effect | Open (euchromatin) | Close (heterochromatin) |
| Gene expression | Activate | Silence |
| Example | p300/CBP, GCN5 | HDAC1, HDAC2, Sir2 |
| Recruited by | Activators | Repressors |
DNA Methylation vs Histone Acetylation
| Feature | DNA Methylation | Histone Acetylation |
|---|---|---|
| Target | CpG dinucleotides (cytosine) | Lysine residues on histone tails |
| Effect on transcription | Silencing | Activation |
| Heritability | Maintained by DNMT1 through replication | Less stable; requires continuous HAT activity |
| Enzymes (writers) | DNMT3a/3b (de novo), DNMT1 (maintenance) | p300/CBP, GCN5 |
| Erasers | TET enzymes (demethylation) | HDACs |
| Stability | Very stable; long-term silencing | More dynamic; rapidly reversible |
miRNA vs siRNA
| Feature | miRNA | siRNA |
|---|---|---|
| Origin | Endogenous; transcribed by Pol II from miRNA genes | Exogenous or endogenous dsRNA; experimental introduction |
| Precursor | pri-miRNA → pre-miRNA hairpin | Long double-stranded RNA |
| Processing | Drosha (nucleus) + Dicer (cytoplasm) | Dicer only (cytoplasm) |
| Base pairing with target | Imperfect (seed region match) | Perfect complementarity |
| Mechanism of silencing | Translational repression + deadenylation | mRNA cleavage (Argonaute/Slicer) |
| Number of targets | Many (~100s of mRNAs per miRNA) | Usually one specific target |
Euchromatin vs Heterochromatin
| Feature | Euchromatin | Heterochromatin |
|---|---|---|
| Appearance | Decondensed, loose | Condensed, compact |
| Transcription | Active | Silent |
| Histone marks | H3K4me3, H3K27ac, H3K36me3 | H3K9me3, H3K27me3 |
| DNA methylation | Low (at CpG islands) | High |
| Replication timing | Early S phase | Late S phase |
| Example | Housekeeping gene promoters | Centromeres (constitutive), inactive X (facultative) |
Diagrams (5)
Enhancer-Mediator Complex
Chromatin States
CpG Methylation
miRNA Pathway
Epigenetic Inheritance
Exam Traps (10)
Research Methods for Transcription Factors
1. EMSA / Gel Shift — "Does protein bind this DNA?"
Readout: Presence/absence of shifted band = binding yes/no. Competition with excess unlabeled DNA confirms specificity.
2. DNase Footprinting — "WHERE on DNA does protein bind?"
Readout: Single-nucleotide resolution of the binding site location on a specific DNA fragment.
3. ChIP (Chromatin Immunoprecipitation) — "What DNA does protein bind IN VIVO?"
Key advantage: Works in native chromatin context with all histone modifications and nucleosome positioning intact — true in vivo binding, not in vitro.
4. Reporter Gene Assay — "Does this DNA region drive transcription?"
Readout: Quantitative measure of transcriptional strength. Mutational analysis pinpoints essential cis-regulatory nucleotides.
Transcription Factor Research Methods — Comparison
Ch8: Protein Synthesis, Processing, and Regulation
Vocabulary (25 terms)
3-nt mRNA unit read 5'→3' during translation. 64 codons: 61 sense (amino acids) + 3 stop (UAA/ochre, UAG/amber, UGA/opal). AUG = start (Met). Code is degenerate (redundant) but unambiguous. Wobble: position 3 of codon tolerates non-Watson-Crick pairing (inosine in tRNA anticodon pairs with U, C, or A)
Key facts: The basic unit of the genetic code; 64 codons total (61 amino acid + 3 stop)
Three-nucleotide sequence on tRNA that base-pairs with complementary mRNA codon
Key facts: The adaptor function of tRNA — matches amino acid to mRNA instruction
20 enzymes (1 per amino acid), each recognizes specific amino acid + all its cognate tRNAs. 2-step reaction: (1) aa + ATP → aminoacyl-AMP + PPi (activation), (2) aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP (transfer to 3'-OH of tRNA acceptor stem CCA). Class I: acylate 2'-OH. Class II: acylate 3'-OH
Key facts: Proofreading: editing site hydrolyzes mischarged aa-tRNA (e.g., IleRS editing site rejects Val). Error rate <10⁻⁴. PPi → 2Pi (pyrophosphatase) drives reaction forward. Net cost: 2 high-energy bonds per aa. tRNA identity elements: acceptor stem + anticodon. Class I synthetases acylate 2′-OH, Class II acylate 3′-OH
Prok 70S = 30S (16S rRNA + 21 proteins) + 50S (23S + 5S rRNA + 31 proteins). Euk 80S = 40S (18S rRNA + ~33 proteins) + 60S (28S + 5.8S + 5S rRNA + ~49 proteins). 3 tRNA sites: A (aminoacyl, incoming), P (peptidyl, growing chain), E (exit, deacylated). Peptidyl transferase = 23S/28S rRNA (ribozyme)
Key facts: Antibiotics target prok 70S: chloramphenicol (blocks peptidyl transferase, 50S), tetracycline (blocks A-site tRNA binding, 30S), erythromycin (blocks exit tunnel, 50S), streptomycin (causes misreading, 30S). Cycloheximide targets euk 80S (blocks translocation). Puromycin = aa-tRNA mimic, causes premature termination (both prok + euk)
Ribosomal site where incoming aminoacyl-tRNA binds, paired with the mRNA codon
Key facts: Entry point for new amino acids; accuracy checked here by decoding center
Ribosomal site holding the tRNA attached to the growing polypeptide chain
Key facts: The 'working bench' where the peptide bond was just formed
Ribosomal site where deacylated (empty) tRNA exits the ribosome
Key facts: Releases spent tRNA back to cytosol for recharging
Prok initiation: AGGAGG consensus, 5-10 nt upstream of AUG. Base-pairs with 3' end of 16S rRNA (anti-Shine-Dalgarno: 3'-AUUCCUCCACUAG-5'). Allows internal initiation → polycistronic mRNAs translated. Absent in eukaryotes (euk use 5' cap scanning)
Key facts: Allows internal initiation on polycistronic mRNAs; absent in eukaryotes
Cap-binding protein in eIF4F complex (eIF4E + eIF4G scaffold + eIF4A helicase). eIF4E binds m7G cap → eIF4G bridges to PABP (circularizes mRNA) + recruits 43S PIC. Rate-limiting for translation initiation
Key facts: 4E-BP sequesters eIF4E when hypophosphorylated → blocks cap-dependent translation. mTOR phosphorylates 4E-BP → releases eIF4E → translation ON. eIF2 regulation: eIF2-GTP delivers Met-tRNAᵢ to 40S; eIF2α phosphorylation (by kinases: HRI=heme, GCN2=amino acids, PERK=ER stress, PKR=dsRNA/viral) → global translation inhibition + selective ATF4 translation. IRE/IRP system: iron low → IRP binds IRE in ferritin mRNA 5'UTR → blocks translation; IRP binds TfR mRNA 3'UTR → stabilizes it
Multiple ribosomes simultaneously translating a single mRNA molecule
Key facts: Allows efficient protein production from one mRNA
Catalytic activity (in large ribosomal subunit rRNA) that forms peptide bonds
Key facts: Ribozyme — rRNA catalyzes the reaction, not protein
Prok: RF1 recognizes UAA + UAG, RF2 recognizes UAA + UGA (GGQ motif mimics tRNA, triggers hydrolysis of peptidyl-tRNA ester bond), RF3 = GTPase recycles RF1/RF2. Euk: eRF1 recognizes all 3 stops (single factor), eRF3 = GTPase. After release: ribosome recycling factor (RRF) + EF-G split 70S → 30S + 50S
Key facts: RF1/RF2 GGQ motif enters peptidyl transferase center, triggers ester bond hydrolysis. Suppressor tRNAs can read through stop codons. Selenocysteine = 21st aa, encoded by UGA + SECIS element in 3'UTR. Puromycin = structural mimic of aminoacyl-tRNA (Tyr), enters A site, forms peptide bond, causes premature termination (used experimentally, targets both prok + euk)
Proteins (Hsp70, Hsp60/GroEL) that assist proper protein folding without being part of the final structure
Key facts: Prevent aggregation; provide a protected environment for folding
Chaperone (DnaK in bacteria). ATP-bound = open lid, low substrate affinity → binds hydrophobic patches. ATP hydrolysis (stimulated by Hsp40/DnaJ co-chaperone) → closed lid, tight grip on substrate. Nucleotide exchange factor (GrpE/BAG1) replaces ADP → substrate released → refolds or transferred to chaperonin
Key facts: Acts co-translationally (ribosome-associated Hsp70/RAC) + post-translationally + during heat shock (HSF1 induces Hsp70 expression). Required for mito/chloro import: matrix Hsp70 (mtHsp70) pulls unfolded polypeptide through TIM23 channel (Brownian ratchet model). DnaK/DnaJ/GrpE = bacterial Hsp70 system
GroEL = 2 stacked 7-mer rings (14 subunits total, each ~57 kDa). GroES = 7-mer co-chaperonin lid. Substrate (~20-60 kDa) binds hydrophobic interior of open ring → GroES caps → 7 ATP bind → conformational change makes cavity hydrophilic → substrate folds inside for ~10 sec → ATP hydrolysis → GroES + substrate released
Key facts: 7 ATP consumed per folding cycle (per ring). Only ~10% of E. coli proteins require GroEL (mostly 20-60 kDa). Euk equivalent = TRiC/CCT (8-subunit ring, no detachable lid). Anfinsen cage model: hydrophilic cavity prevents aggregation, allows single-molecule folding
76-aa protein (8.5 kDa), highly conserved. Conjugated to substrate Lys via isopeptide bond (C-terminal Gly). Pathway: E1 (ubiquitin-activating, 2 in humans, uses ATP) → E2 (conjugating, ~40 types) → E3 (ligase, ~600+ types, provides substrate specificity) → polyUb chain
Key facts: K48-linked polyUb (≥4 Ub) = proteasomal degradation. K63-linked = signaling (NF-κB, DNA repair). Mono-Ub = endocytic sorting, histone regulation (H2BK120ub). 26S proteasome = 20S core (4 stacked 7-mer rings: α7β7β7α7; β1/β2/β5 = proteolytic active sites) + 19S regulatory particle (recognizes polyUb, has ATPases to unfold + feed substrate through α-ring gate). DUBs (deubiquitinases) recycle Ub before substrate enters 20S
Large barrel-shaped protease complex that degrades ubiquitylated proteins to peptides
Key facts: Regulated protein destruction; requires ATP; recycles ubiquitin
Ubiquitin-activating (E1), conjugating (E2), and ligase (E3) enzymes in the ubiquitin pathway
Key facts: E3 provides substrate specificity — determines WHICH proteins get ubiquitylated
Process where cells degrade their own organelles/proteins by enclosing them in autophagosomes that fuse with lysosomes
Key facts: Bulk degradation for recycling during starvation or for quality control
System where protein half-life is determined by its N-terminal amino acid (N-end rule)
Key facts: Destabilizing N-terminal residues target proteins for rapid ubiquitylation
N-terminal ~16-30 aa: positively charged n-region + hydrophobic h-region (core, 7-15 hydrophobic aa) + polar c-region (signal peptidase cleavage site). Recognized co-translationally by SRP (signal recognition particle) when it emerges from ribosome exit tunnel
Key facts: SRP = 6 proteins + 7SL RNA (in mammals). SRP binds signal peptide emerging from ribosome exit tunnel → pauses translation → docks at SRP receptor (SRα/SRβ, GTPases) on ER membrane → ribosome-nascent chain transferred to Sec61 translocon → translation resumes co-translationally → signal peptidase cleaves signal in ER lumen. Type II membrane proteins use signal-anchor (NOT cleaved)
Covalent changes to proteins after synthesis: phosphorylation, glycosylation, acetylation, etc.
Key facts: Regulate protein activity, localization, interactions, and stability
Enzyme that transfers phosphate from ATP to serine/threonine/tyrosine residues on proteins
Key facts: Master regulators of cell signaling; ~500 kinases in human genome
Enzyme that removes phosphate groups from proteins, reversing kinase action
Key facts: Works opposite to kinases; together they create reversible signaling switches
3rd codon position tolerates non-Watson-Crick pairing at anticodon position 1: G-U pairs allowed; inosine (I) in anticodon pairs with U, C, or A → one tRNA reads up to 3 codons. Minimum 31 tRNAs needed (not 61) to decode all sense codons.
Key facts: Explains why only ~45 tRNAs can read all 61 sense codons; inosine (I) in anticodon pairs with U, C, or A
Mechanisms (5)
Eukaryotic Translation Initiation
Translation Elongation Cycle
Protein Folding by Chaperones: Hsp70 → Hsp60
Ubiquitin-Proteasome Degradation Pathway
Autophagy
Comparison Tables (4)
Prokaryotic vs Eukaryotic Translation
| Feature | Prokaryotic | Eukaryotic |
|---|---|---|
| Ribosome size | 70S (50S + 30S) | 80S (60S + 40S) |
| Initiation | Shine-Dalgarno + 16S rRNA; fMet-tRNA | 5' cap scanning; Met-tRNA; 12+ eIFs |
| Start amino acid | N-formylmethionine | Methionine (unmodified) |
| mRNA type | Polycistronic (multiple ORFs) | Monocistronic (one ORF) |
| Coupling | Transcription-translation coupled | Separated by nuclear envelope |
| Elongation factors | EF-Tu, EF-G | eEF1α, eEF2 |
Hsp70 vs Hsp60 Chaperones
| Feature | Hsp70 | Hsp60 (GroEL/GroES) |
|---|---|---|
| Structure | Monomeric clamp | Barrel-shaped cage (double ring) |
| Binding mode | Binds exposed hydrophobic stretches | Encapsulates entire protein in chamber |
| When it acts | Co-translationally + early folding | Post-translational; after Hsp70 |
| Energy | ATP hydrolysis (open/close cycle) | ATP hydrolysis (conformational change) |
| Substrate size | Binds small peptide segments | Proteins up to ~60 kDa fit in barrel |
| Co-chaperones | Hsp40 (J-domain) | GroES (lid) |
Ubiquitin-Proteasome vs Autophagy
| Feature | Ubiquitin-Proteasome | Autophagy |
|---|---|---|
| Substrates | Individual proteins | Bulk: proteins, organelles, aggregates |
| Selectivity | Highly selective (E3 specificity) | Can be selective or non-selective |
| Marker | K48-polyubiquitin chain | LC3/p62 for selective; none for bulk |
| Degradation site | Proteasome (cytosolic) | Lysosome (acidic compartment) |
| Energy cost | ATP for ubiquitylation + unfolding | ATP for membrane assembly |
| Trigger | Specific degrons/signals | Starvation, stress, damaged organelles |
| Products | Peptides (7-9 residues) | Amino acids, fatty acids, sugars |
Co-translational vs Post-translational Modifications
| Feature | Co-translational | Post-translational |
|---|---|---|
| Timing | During translation | After translation complete |
| Examples | Signal peptide cleavage, N-glycosylation, disulfide bonds (in ER) | Phosphorylation, ubiquitylation, proteolytic cleavage |
| Location | Ribosome/ER lumen | Cytosol, ER, Golgi, etc. |
| Reversibility | Usually irreversible | Often reversible (phosphorylation) |
| Purpose | Targeting, initial folding | Regulation, activation, degradation |
Diagrams (5)
Ribosome A/P/E Sites
Translation Elongation
Chaperone Folding Pathway
Ubiquitin-Proteasome Pathway
Post-translational Modifications
Exam Traps (10)
Ch11: Protein Sorting and Transport
Vocabulary (22 terms)
~16-30 aa N-terminal peptide: positively charged n-region → hydrophobic h-region (7-15 Leu/Ala, forms α-helix in Sec61 channel) → polar c-region with signal peptidase cleavage site (Ala-X-Ala rule)
Key facts: Recognized co-translationally by SRP54 methionine-rich M domain; inserts into Sec61 translocon lateral gate; cleaved by signal peptidase on ER lumenal side
Ribonucleoprotein (6 proteins + SRP RNA) that binds signal sequence and pauses translation
Key facts: Molecular escort: bridges ribosome to ER membrane
ER membrane protein that binds SRP-ribosome complex; triggers GTP hydrolysis to release SRP
Key facts: Docking station on the ER for incoming ribosomes
Protein channel (3 subunits: α,β,γ) in ER membrane through which polypeptides are threaded
Key facts: Has a plug that opens when signal sequence inserts; lateral gate for transmembrane domains
ER lumenal enzyme that cleaves the signal sequence from translocating proteins
Key facts: Removes the 'address label' once delivery is confirmed
Hydrophobic transmembrane domain that halts translocation and anchors protein in ER membrane
Key facts: Determines single-pass membrane protein topology
ER-resident chaperone that assists protein folding in the ER lumen
Key facts: Binds unfolded proteins as they enter; also sensor for UPR
Addition of 14-sugar oligosaccharide to asparagine (Asn-X-Ser/Thr) in ER from dolichol carrier
Key facts: Aids folding, prevents aggregation, provides quality control signals
Addition of sugars to serine/threonine hydroxyl groups; occurs in the Golgi
Key facts: Further modification for cell surface and secreted proteins
Coat protein complex II; mediates anterograde transport (ER → Golgi)
Key facts: Selective: concentrates cargo proteins with ER exit signals
Coat protein complex I; mediates retrograde transport (Golgi → ER)
Key facts: Retrieves escaped ER-resident proteins (recognize KDEL signal)
Vesicles coated with clathrin triskelion; mediate transport from Golgi/plasma membrane
Key facts: Used in receptor-mediated endocytosis and M6P lysosomal sorting
Lys-Asp-Glu-Leu C-terminal sequence that marks ER-resident soluble proteins for retrieval from Golgi
Key facts: KDEL receptor in Golgi captures escaped proteins and returns them via COPI
Phosphorylated mannose residue added to lysosomal enzymes in cis-Golgi; recognized by M6P receptor
Key facts: The 'lysosome address tag'; recognized by M6P receptor in trans-Golgi for sorting
v-SNAREs (on vesicle) and t-SNAREs (on target) that mediate membrane fusion via coiled-coil zippering
Key facts: Ensure vesicles fuse with correct target membrane; specificity mechanism
Small GTPases on vesicle surfaces that regulate vesicle targeting and tethering
Key facts: Act as molecular switches: GTP-bound = active, GDP-bound = inactive
Quality control: misfolded ER proteins retrotranslocated to cytosol and degraded by proteasome
Key facts: Prevents accumulation of defective proteins in the secretory pathway
Signaling pathway activated when misfolded proteins accumulate in ER; upregulates chaperones
Key facts: Adaptive response: increases ER folding capacity; if overwhelmed → apoptosis
Default pathway: continuous vesicle fusion with plasma membrane without specific signal
Key facts: How most membrane proteins and ECM components reach the surface
Signal-triggered release of stored secretory vesicles (e.g., neurotransmitters, hormones)
Key facts: Requires external stimulus (Ca²⁺, hormone); contents stored in dense-core granules
Glycosylphosphatidylinositol lipid anchor attached to C-terminus; tethers protein to outer membrane leaflet
Key facts: Alternative to transmembrane domain; assembled in ER, added to protein post-translationally
ER enzyme that catalyzes formation and rearrangement of disulfide bonds
Key facts: Oxidizing ER environment (unlike reducing cytosol) enables S-S bond formation
Lipid Synthesis in the ER and Golgi
Phospholipid Synthesis — Smooth ER, Cytosolic Leaflet
Key fact: Smooth ER is the primary site of phospholipid synthesis. Lipids must be redistributed to other organelle membranes via vesicular transport or lipid transfer proteins.
Flippases vs Scramblases — Membrane Asymmetry
Key fact: PS on outer leaflet also used clinically — Annexin V binds PS and is used to detect apoptotic cells by flow cytometry.
Cholesterol and Sphingolipid Synthesis
Lipid Synthesis: ER → Golgi Pathway
Mechanisms (7)
SRP-Mediated Co-translational Translocation into ER
Stop-Transfer and Signal-Anchor Membrane Insertion
N-linked Glycosylation in ER → O-linked in Golgi
COPII (ER→Golgi) vs COPI (Golgi→ER) Vesicle Transport
Mannose-6-Phosphate Lysosomal Targeting
Clathrin-Mediated Endocytosis
Unfolded Protein Response (UPR) — Three Signaling Arms
Methods of Investigating Sorting and Transport
1. Pulse-Chase — "What ORDER and TIMING?"
Key insight: Pulse-chase reveals the sequential order and kinetics of compartment transit. Protein stuck at a particular step = that step requires the mutated gene product.
2. Cell-Free Reconstitution Assay (Rothman) — "What MOLECULES?"
Key insight: Nobel Prize 2013 (Rothman, Schekman, Südhof) for discoveries of machinery regulating vesicle traffic. Cell-free assays identify the specific molecules required.
3. Yeast sec Mutants (Schekman) — "What GENES?"
Key insight: Accumulation site = step blocked. ER accumulation = coat assembly defect. Vesicle accumulation = fusion machinery defect.
4. GFP Tagging & FRAP — "WHERE in Live Cells?"
Key insight: GFP tracks localization and dynamics in living cells. FRAP quantifies transport rates and protein mobility.
5. Brefeldin A (BFA) — "What If COPI Is Blocked?"
Key insight: BFA proves the Golgi is a dynamic steady-state organelle maintained by continuous vesicle traffic, not a static structure. COPI retrograde flow is required for Golgi identity.
Methods for Investigating Sorting & Transport
Comparison Tables (5)
Co-translational vs Post-translational Translocation
| Feature | Co-translational | Post-translational |
|---|---|---|
| Timing | During translation | After translation complete |
| Targeting | SRP → SRP receptor | Cytosolic chaperones (Hsp70/Hsc70) |
| Channel | Sec61 (ribosome-bound) | Sec61 or Sec62/63 (ribosome-free) |
| Organisms | Predominant in mammals | Common in yeast; some mammalian proteins |
| Energy | Translation itself drives translocation | BiP/Hsp70 ATP-dependent pulling |
| Folding state | Unfolded (being synthesized) | Must be kept unfolded by chaperones |
COPI vs COPII vs Clathrin Vesicles
| Feature | COPII | COPI | Clathrin |
|---|---|---|---|
| Direction | ER → Golgi (anterograde) | Golgi → ER (retrograde) | TGN → endosome/PM; PM → endosome |
| GTPase | Sar1 | ARF1 | ARF1 (TGN) / dynamin (PM) |
| Coat proteins | Sec23/24, Sec13/31 | Coatomer (α-ζ COPs) | Clathrin + adaptors (AP1, AP2) |
| Cargo signal | ER exit signals (di-acidic, di-hydrophobic) | KDEL, KKXX (ER retrieval) | Tyrosine/dileucine motifs in receptors |
| Main function | Forward transport of cargo proteins | Retrieve ER residents from Golgi | Lysosomal sorting; endocytosis |
Constitutive vs Regulated Secretion
| Feature | Constitutive | Regulated |
|---|---|---|
| Signal required? | No — continuous | Yes — external trigger (Ca²⁺, etc.) |
| Vesicle storage | No storage; immediate fusion | Stored in secretory granules |
| Cell types | All cells | Specialized cells (neurons, endocrine, exocrine) |
| Cargo examples | ECM proteins, membrane proteins | Neurotransmitters, hormones, digestive enzymes |
| Coat protein | COPII → default pathway | Clathrin from TGN → stored granules |
Signal Sequence vs Signal Patch
| Feature | Signal Sequence | Signal Patch |
|---|---|---|
| Nature | Linear, continuous amino acid sequence | 3D arrangement of residues from different parts of chain |
| Location | Usually N-terminal | Scattered across protein; only formed when folded |
| Cleavable? | Usually yes (by signal peptidase) | No — part of mature protein structure |
| Example | ER signal peptide, mitochondrial presequence | M6P lysosomal targeting signal, nuclear localization signal |
| Recognition | Recognized in unfolded state (as it exits ribosome) | Recognized in folded state |
ER vs Golgi Glycosylation
| Feature | ER Glycosylation | Golgi Glycosylation |
|---|---|---|
| Type | N-linked (Asn-X-Ser/Thr) | N-linked modification + O-linked (Ser/Thr) |
| Mechanism | Block transfer (14 sugars from dolichol) | Sequential addition by glycosyltransferases |
| Sugars added | GlcNAc, mannose, glucose | GlcNAc, galactose, sialic acid, fucose |
| Processing | Trimming of glucose and some mannose | Trimming + extensive addition |
| Quality control role | Yes (calnexin/calreticulin cycle) | No QC role — final processing |
Diagrams (6)
Co-translational Translocation
Membrane Protein Topologies
Secretory Pathway
COPII/COPI/Clathrin
M6P Lysosomal Sorting
Clathrin-Mediated Endocytosis
Exam Traps (10)
Ch12: Mitochondria, Chloroplasts, and Peroxisomes
Vocabulary (26 terms)
Double-membrane organelle; site of oxidative phosphorylation and ATP production
Key facts: The cell's power plant; contains own genome; evolved from α-proteobacteria
Folds of the mitochondrial inner membrane that increase surface area for oxidative phosphorylation
Key facts: More cristae = more ETC complexes = more ATP production capacity
Inner compartment containing citric acid cycle enzymes, mtDNA, ribosomes
Key facts: The working interior of mitochondria where pyruvate and fatty acids are oxidized
Space between inner and outer mitochondrial membranes; similar composition to cytosol
Key facts: Where protons accumulate during electron transport → pH lower than matrix
Channel proteins in mitochondrial outer membrane allowing free diffusion of molecules <1000 Da
Key facts: Makes outer membrane freely permeable to small molecules (unlike inner membrane)
Series of protein complexes (I-IV) in inner membrane that transfer electrons from NADH/FADH₂ to O₂
Key facts: Couples electron transfer to proton pumping across inner membrane
Mitchell's theory: proton gradient across inner membrane drives ATP synthesis by ATP synthase
Key facts: Electrochemical gradient (ΔΨ + ΔpH) = proton-motive force
Rotary enzyme in inner membrane that synthesizes ATP from ADP + Pi using proton flow
Key facts: F₀ portion = proton channel in membrane; F₁ = catalytic head in matrix
Combined electrochemical potential (voltage + pH gradient) across inner mitochondrial membrane
Key facts: Drives both ATP synthesis and metabolite transport
N-terminal targeting signal (15-55 aa, positively charged amphipathic helix) for mitochondrial import
Key facts: Removed by matrix processing peptidase (MPP) after import
Translocase of the Outer Membrane; general entry point for all mitochondrial protein import
Key facts: Receptor subunits recognize presequences and internal signals
Translocase of the Inner Membrane; imports presequence-containing proteins to matrix or inner membrane
Key facts: Driven by membrane potential (negative inside attracts positive presequence)
Second inner membrane translocase; inserts multi-pass carrier proteins with internal signals
Key facts: Uses Tim9-Tim10 chaperones in IMS for delivery
Small chaperones in intermembrane space that escort hydrophobic proteins between TOM and TIM22
Key facts: Prevent aggregation of hydrophobic inner membrane proteins in aqueous IMS
Protease in mitochondrial matrix that cleaves the presequence after protein import
Key facts: Removes the 'address label' once the protein reaches its destination
Sorting and Assembly Machinery in outer membrane; inserts β-barrel proteins
Key facts: After proteins pass through TOM, SAM inserts them into the outer membrane
Double-membrane + thylakoid membrane organelle; site of photosynthesis in plants
Key facts: Three membranes → three compartments (IMS, stroma, thylakoid lumen)
Internal membrane system of chloroplasts; contains photosystems and ATP synthase for light reactions
Key facts: Where light energy is captured and converted to chemical energy
Aqueous interior of chloroplast (outside thylakoids); contains Calvin cycle enzymes and chloroplast DNA
Key facts: Equivalent to mitochondrial matrix; site of carbon fixation
Translocase of Outer/Inner Chloroplast membrane; imports nuclear-encoded chloroplast proteins
Key facts: Transit peptides (not presequences) target proteins; require GTP + ATP
N-terminal targeting signal for chloroplast import; cleaved by stromal processing peptidase
Key facts: Differs from mito presequence: not positively charged amphipathic helix
Mitochondria evolved from α-proteobacteria; chloroplasts from cyanobacteria engulfed by ancestral eukaryote
Key facts: Supported by: own DNA, double membrane, bacterial-like ribosomes, binary fission
Single-membrane organelle containing oxidative enzymes (catalase, oxidases); involved in fatty acid β-oxidation
Key facts: Generates H₂O₂ as byproduct; catalase converts it to H₂O + O₂
Genes encoding peroxin proteins required for peroxisome biogenesis and protein import
Key facts: Mutations cause Zellweger syndrome (peroxisome biogenesis disorder)
C-terminal tripeptide (SKL or conservative variant) that targets most matrix proteins to peroxisomes
Key facts: Recognized by Pex5 receptor in cytosol; most common peroxisomal import signal
N-terminal nonapeptide signal targeting a few peroxisomal matrix proteins; recognized by Pex7 receptor
Key facts: Only used by a few proteins (e.g., thiolase); cleaved after import in some organisms
Mechanisms (7)
Mitochondrial Protein Import via TOM/TIM23
TIM22 Pathway (Inner Membrane Carrier Proteins)
Electron Transport Chain (Complexes I-IV)
Chemiosmotic Coupling (ATP Synthesis)
Chloroplast Protein Import (TOC/TIC)
Light Reactions of Photosynthesis
Peroxisome Protein Import (PTS1/PTS2 Pathways)
Comparison Tables (5)
Mitochondria vs Chloroplasts
| Feature | Mitochondria | Chloroplasts |
|---|---|---|
| Membranes | 2 (outer + inner) | 3 (outer + inner + thylakoid) |
| Compartments | 4 (OM, IMS, IM, matrix) | 3 + thylakoid lumen |
| Energy process | Oxidative phosphorylation | Photosynthesis (+ some oxidative) |
| Inputs | O₂, NADH, FADH₂, ADP | Light, H₂O, CO₂, ADP, NADP⁺ |
| Outputs | ATP, H₂O, CO₂ | ATP, NADPH, O₂, G3P |
| Genome size (human/plant) | ~16 kb / ~370 kb | N/A / ~120-150 kb |
| Endosymbiont ancestor | α-proteobacterium | Cyanobacterium |
| Protein import signal | Presequence (positively charged) | Transit peptide (not positively charged) |
| Import translocases | TOM/TIM23/TIM22 | TOC/TIC |
TOM/TIM23 vs TOM/TIM22
| Feature | TOM/TIM23 pathway | TOM/TIM22 pathway |
|---|---|---|
| Targeting signal | N-terminal presequence (cleavable, positively charged) | Internal signals (non-cleavable) |
| Substrates | Matrix proteins, some inner membrane proteins | Multi-pass inner membrane carriers |
| IMS chaperones | Not needed (direct transfer) | Tim9-Tim10 required |
| Driving force | Membrane potential + matrix Hsp70 motor | Membrane potential |
| Signal cleavage | Yes (MPP in matrix) | No (internal signals retained) |
| Example cargo | Citric acid cycle enzymes | ADP/ATP translocator, phosphate carrier |
Oxidative Phosphorylation vs Photophosphorylation
| Feature | Oxidative (Mitochondria) | Photo (Chloroplasts) |
|---|---|---|
| Energy source | Chemical (NADH, FADH₂) | Light |
| Electron donor | NADH, FADH₂ | H₂O |
| Final electron acceptor | O₂ → H₂O | NADP⁺ → NADPH |
| Location | Inner mitochondrial membrane | Thylakoid membrane |
| H⁺ gradient direction | Matrix → IMS (matrix alkaline) | Stroma → thylakoid lumen (lumen acidic) |
| ATP made in | Matrix | Stroma |
Presequence vs Transit Peptide
| Feature | Mitochondrial Presequence | Chloroplast Transit Peptide |
|---|---|---|
| Charge | Positively charged (Arg, Lys) | Not strongly charged; no amphipathic helix |
| Structure | Amphipathic α-helix | No clear secondary structure |
| Length | 15-55 amino acids | 20-80+ amino acids |
| Cleavage | MPP in matrix | SPP in stroma |
| Driving force for import | Electrochemical gradient (ΔΨ) + Hsp70 motor | GTP (TOC) + ATP (Hsp70/93 in stroma) |
| Translocases | TOM → TIM23 | TOC → TIC |
Matrix Targeting vs Inner Membrane Insertion
| Feature | Matrix targeting | Inner membrane insertion |
|---|---|---|
| Signal | Presequence only | Presequence + stop-transfer OR internal signals |
| Pathway | TOM → TIM23 → matrix | TOM → TIM23 (lateral exit) OR TOM → TIM22 |
| Presequence cleaved? | Yes, by MPP | Depends: TIM23 path = yes; TIM22 path = no |
| Final location | Soluble in matrix | Embedded in inner membrane |
| Examples | Citric acid cycle enzymes, mtHsp70 | ETC complexes, metabolite carriers |
Diagrams (6)
Mitochondrion Structure
TOM/TIM Import
Electron Transport Chain
Chloroplast Structure
Endosymbiotic Origin
Peroxisome Protein Import
Exam Traps (10)
Cross-Chapter Patterns
| Pattern | How It Appears Across Chapters |
|---|---|
| Signal-directed targeting | Ch6: promoter sequences direct Pol to genes | Ch7: enhancer sequences direct activators | Ch8: Shine-Dalgarno/5' cap directs ribosomes | Ch11: signal sequence → ER; M6P → lysosome | Ch12: presequence → mito matrix; transit peptide → chloroplast |
| Energy cost | Ch6: NTP hydrolysis for transcription | Ch7: ATP for chromatin remodeling | Ch8: 2 GTP per amino acid in elongation + ATP for chaperones | Ch11: GTP for SRP/SNARE/coat assembly | Ch12: ΔΨ + ATP for mito import; ATP for chloroplast import |
| Quality control | Ch6: RNA surveillance/degradation of aberrant mRNAs | Ch7: histone code readers verify chromatin state | Ch8: ribosome proofreading + chaperone folding + ubiquitin-proteasome | Ch11: ERAD + UPR in ER; calnexin/calreticulin cycle | Ch12: mitochondrial proteases degrade misfolded matrix proteins |
| Prokaryotic vs eukaryotic | Ch6: one RNA Pol vs three; σ vs GTFs; no processing vs extensive processing | Ch7: operons vs enhancers/chromatin | Ch8: 70S vs 80S; SD vs cap-scanning; fMet vs Met | Ch11: Sec pathway in bacteria ≈ SRP/Sec61 in eukaryotes | Ch12: bacterial membranes ≈ mito inner membrane (endosymbiosis) |
| Co-translational vs post-translational | Ch6: mRNA capping/splicing are co-transcriptional | Ch8: co-translational folding by ribosome-associated Hsp70 | Ch11: co-translational translocation into ER (SRP) vs post-translational import | Ch12: post-translational import into mito/chloroplasts |
| Enzyme-substrate specificity | Ch6: σ factor selects promoters; snoRNPs guide rRNA modification sites | Ch7: E3 ubiquitin ligases determine which TFs are degraded | Ch8: aminoacyl-tRNA synthetases (1 per amino acid); E3 ligases | Ch11: M6P receptor binds specifically M6P-tagged enzymes | Ch12: TOM receptors distinguish presequences from internal signals |
| Regulated vs constitutive | Ch6: housekeeping gene promoters (constitutive) vs inducible promoters | Ch7: constitutive heterochromatin vs facultative | Ch8: global translation vs mRNA-specific regulation by miRNAs | Ch11: constitutive vs regulated secretion | Ch12: constitutive mitochondrial biogenesis vs stress-induced mitophagy |
| Disease connection | Ch6: splicing defects → Duchenne muscular dystrophy (exon skipping therapy) | Ch7: epigenetic misregulation → cancer (HDAC inhibitors as therapy) | Ch8: protein misfolding → Alzheimer's (Aβ aggregation) | Ch11: I-cell disease (M6P defect); Gaucher disease (lysosomal enzyme deficiency) | Ch12: LHON (mtDNA mutations in ETC); Zellweger syndrome (peroxisome biogenesis) |
Cross-Chapter Comparisons
Transcription vs Translation
| Feature | Transcription | Translation |
|---|---|---|
| Template | DNA (template strand) | mRNA |
| Product | RNA | Protein |
| Enzyme | RNA polymerase | Ribosome (rRNA = ribozyme) |
| Direction | 5'→3' RNA synthesis | 5'→3' mRNA reading; N→C protein synthesis |
| Initiation signals | Promoter (TATA box, -10/-35) | Start codon (AUG) + Shine-Dalgarno or 5' cap |
| Termination | Terminator sequence / poly-A signal | Stop codon (UAA, UAG, UGA) |
| Energy | NTPs | GTP (elongation factors) + ATP (tRNA charging) |
mRNA Processing vs Protein Processing
| Feature | mRNA Processing | Protein Processing |
|---|---|---|
| Location | Nucleus | ER, Golgi, cytosol |
| 5' modification | 7-methylguanosine cap | Signal peptide cleavage |
| 3' modification | Poly-A tail addition | GPI anchor addition |
| Internal modification | Splicing (intron removal) | Glycosylation, disulfide bonds, folding |
| Quality control | Nonsense-mediated decay | ERAD, chaperone-assisted folding |
| Regulation | Alternative splicing | Phosphorylation, ubiquitylation |
Signal Sequence vs Presequence vs Transit Peptide
| Feature | ER Signal Sequence | Mito Presequence | Chloroplast Transit Peptide |
|---|---|---|---|
| Destination | ER lumen/membrane | Mito matrix/inner membrane | Chloroplast stroma/thylakoid |
| Character | Hydrophobic | Positively charged amphipathic helix | No strong charge pattern |
| Length | 15-40 aa | 15-55 aa | 20-80+ aa |
| When recognized | Co-translationally (SRP) | Post-translationally (Hsp70) | Post-translationally |
| Cleavage | Signal peptidase (in ER) | MPP (in matrix) | SPP (in stroma) |
| Energy for import | Translation (GTP) | ΔΨ + ATP (Hsp70 motor) | GTP + ATP |
| Translocase | Sec61 translocon | TOM → TIM23 | TOC → TIC |
ER Quality Control (ERAD) vs Cytoplasmic Proteasome
| Feature | ERAD | Cytoplasmic Proteasome |
|---|---|---|
| Substrates | Misfolded ER proteins | Ubiquitylated cytosolic/nuclear proteins |
| Detection | Calnexin/calreticulin cycle; BiP | E3 ligases recognize degrons |
| Key step | Retrotranslocation to cytosol | Direct recognition + unfolding |
| Ubiquitylation | At ER membrane during retrotranslocation | In cytosol by E1/E2/E3 |
| Degradation by | 26S proteasome (in cytosol) | 26S proteasome (in cytosol) |
| Both use proteasome? | Yes — ERAD feeds into proteasomal degradation | Yes |
Vesicular Transport vs Direct Translocation
| Feature | Vesicular Transport | Direct Translocation |
|---|---|---|
| Mechanism | Budding → transport → fusion | Threading through protein channel |
| Examples | ER→Golgi (COPII), endocytosis (clathrin) | ER import (Sec61), mito import (TOM/TIM) |
| Coat proteins | COPI, COPII, clathrin | None (translocon complexes instead) |
| Cargo state | Folded proteins in lumen/membrane | Unfolded polypeptide chains |
| Energy | GTP for coat assembly + SNAREs | Translation / ΔΨ + ATP / GTP |
Mito vs Chloroplast vs Peroxisome Protein Import
| Feature | Mitochondria | Chloroplasts | Peroxisomes |
|---|---|---|---|
| Targeting signal | Presequence (positively charged) | Transit peptide | PTS1 (C-terminal SKL) or PTS2 |
| Signal cleaved? | Yes (MPP) | Yes (SPP) | PTS1: no; PTS2: sometimes |
| Translocases | TOM/TIM23/TIM22/SAM | TOC/TIC | PEX5/PEX7 receptors + importomer |
| Protein folding state | Must be unfolded | Must be unfolded | Can import FOLDED proteins |
| Energy source | ΔΨ + ATP | GTP + ATP | ATP |
| Endosymbiotic origin? | Yes (α-proteobacterium) | Yes (cyanobacterium) | No |