Transcription in Eukaryotes

Transcription in Eukaryotes

Torpedo model of termination involves

Torpedo model explains Pol II termination via cooperation between cleavage and exonucleolytic degradation. After endonucleolytic cut at polyadenylation site, downstream nascent RNA attached to elongating polymerase bears exposed five prime monophosphate lacking cap. Nuclear exonuclease Xrn2 in mammals, Rat1 in yeast, associates with Serine two phosphorylated CTD via Rtt103 and Pcf11, binds uncapped end, degrades RNA processively in five prime to three prime direction. Kinetic chase overtakes polymerase, destabilizes RNA-DNA hybrid and induces allosteric release of polymerase from template, completing termination and allowing recycling.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10: Torpedo model 5'-3' exonuclease Xrn2; Nature Struct Mol Biol, Mechanism of Pol II termination

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Transcription in Eukaryotes

Termination of RNA pol II transcription occurs

Termination of RNA polymerase II transcription is coupled to three prime end processing and occurs after recognition of polyadenylation signal AAUAAA. CPSF-CstF machinery cleaves nascent RNA downstream of signal while polymerase continues transcribing one to two kilobases beyond cleavage site. Uncapped five prime monophosphate of downstream fragment becomes substrate for nuclear 5' to 3' exonuclease Xrn2 Rat1 torpedo that degrades RNA faster than elongation, eventually catching polymerase and triggering dissociation. Allosteric model also proposes conformational change after polyadenylation signal causing loss of elongation factors.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10: Pol II termination after polyadenylation signal; Alberts et al., Torpedo model of termination

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Transcription in Eukaryotes

Poly(A) tail is added by

After cleavage at polyadenylation site, polyadenine tail is synthesized not by RNA polymerase II but by specialized nuclear polyadenine polymerase PAPOLA and PAPOLG, recruited through interaction with CPSF via FIP1 subunit and stimulated by nuclear polyadenine binding protein PABPN1. Enzyme is template independent, uses ATP to add adenylates processively: initial distributive addition of ten to twelve adenines, then PABPN1-dependent switch to processive synthesis of two hundred to two hundred fifty residues in mammals. Polyadenine tail enhances stability, export, and translation via PABPC and eIF4G circularization bridge.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 6: Poly(A) polymerase adds tail; Lodish 9th ed., Mechanism of polyadenylation

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Transcription in Eukaryotes

Polyadenylation signal in eukaryotic mRNA is

Eukaryotic pre-mRNAs contain conserved hexamer AAUAAA twenty to thirty nucleotides upstream of cleavage site, transcribed into RNA as polyadenylation signal essential for three prime end formation. Cleavage and polyadenylation specificity factor CPSF binds AAUAAA via WDR33 and CPSF30 zinc fingers recognizing sequence via base stacking interactions, while cleavage stimulation factor CstF binds downstream GU-rich downstream sequence element. Polyadenylation factors CFI, CFII, and endonuclease CPSF73 catalyze endonucleolytic cut. Signal AAUAAA is essential: single point mutation drastically reduces processing. Similar DNA sequence AATAAA in coding strand is classic Hogness box.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10: Polyadenylation signal AAUAAA recognition by CPSF; Alberts 7th ed., 3' end formation signals

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Transcription in Eukaryotes

5' cap contains which unusual linkage?

Eukaryotic five prime cap harbors highly unusual five prime to five prime linkage instead of canonical three prime to five prime phosphodiester found in RNA backbone. After RNA triphosphatase generates diphosphate RNA, guanylyltransferase hydrolyzes GTP to GMP and catalyzes attack of diphosphate RNA on alpha phosphate forming Gp pppN triphosphate bridge linking guanine five prime to first nucleotide five prime. Three phosphate groups create 5'-5' connection. Subsequent methylation at N7 of guanine and ribose 2' O positions generate cap zero, cap one, cap two structures resistant to decapping and exonuclease attack.

Ref: Berg et al., Biochemistry, 9th ed., Chapter 28: 5'-5' triphosphate cap linkage; Lodish 9th ed., Mechanism of guanylyltransferase forms unusual linkage

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Transcription in Eukaryotes

RNA capping occurs at

Five prime capping occurs at five prime end of nascent pre-mRNA soon after emergence of twenty to thirty nucleotides from exit channel of RNA polymerase II. Mammalian capping enzyme triphosphatase removes gamma phosphate, guanylyltransferase attaches GMP via unusual five prime to five prime triphosphate bridge, and N7-methyltransferase methylates guanine using SAM. Reaction requires serine five phosphorylated CTD that recruits capping enzyme to promoter proximal complex. Cap protects transcript from exonucleolytic degradation, promotes nuclear export via cap binding complex, and enhances translation initiation through eIF4E recognition and circularization.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 6: 5' capping occurs cotranscriptionally; Lodish 9th ed., Capping enzymes and CTD recruitment

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Transcription in Eukaryotes

SPT5 is analogous to which bacterial elongation factor?

SPT5, together with SPT4 forms DSIF transcription elongation complex in eukaryotes, regulating pause release near promoters. Its bacterial counterpart NusG shares conserved NGN domain that contacts clamp domain of RNA polymerase and KOW domains that bind nascent RNA or ribosome. Both factors increase processivity, suppress backtracking, and coordinate transcription with co-transcriptional events: NusG in bacteria bridging RNA polymerase to ribosome or Rho terminator, SPT5 in eukaryotes to chromatin modification machinery and capping. SPT5 C-terminal repeats phosphorylated by P-TEFb CDK9 analogous to Pol II CTD.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10: SPT5 analogous to bacterial NusG; Alberts 7th ed., Conserved elongation factor NusG homology

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Transcription in Eukaryotes

FACT facilitates transcription by

Nucleosome core particle contains one hundred forty-seven base pairs of DNA wrapped around octamer of histones with central H3-H4 tetramer and two peripheral H2A-H2B dimers. To transcribe through chromatin, RNA polymerase II needs partial disassembly. FACT binds to nucleosome and destabilizes dimer-tetramer contacts, causing transient eviction of one H2A-H2B dimer converting nucleosome to hexasome, unwrapping approximately thirty base pairs of entry DNA. This reduction lowers mechanical barrier, decreasing polymerase crossing time measured by optical tweezers. FACT then acts as chaperone to redeposit dimer after passage.

Ref: PubMed 2003 FACT removes H2A-H2B dimer during Pol II passage; Nature 2020 Structural visualization of FACT-mediated nucleosome reorganization

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Transcription in Eukaryotes

FACT complex is involved during

FACT, facilitates chromatin transcription, is heterodimeric histone chaperone composed of SPT16 and SSRP1 in humans, Spt16-Pob3 plus Nhp6 in yeast, that remains associated with elongating Pol II during gene body transcription. Nucleosomes present strong barrier to polymerase progression, causing frequent pausing and backtracking requiring rescue. FACT binds both H2A-H2B dimers and H3-H4 tetramers simultaneously via acidic domains, destabilizing nucleosome without displacing octamer completely, allowing passage. Interaction with SPT5 DSIF complex and PAF1 complex tethers FACT to elongating polymerase ensuring rapid reassembly behind transcription machinery.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10: FACT complex in elongation; JBC, FACT facilitates transcription-dependent nucleosome alteration

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Transcription in Eukaryotes

Sensitivity of RNA polymerases to α-amanitin is

Sensitivity to alpha-amanitin toxin distinguishes three nuclear polymerases and is exploited in nuclear run-on assays. Pol II shows highest sensitivity, IC50 approximately 0.01 microgram per milliliter, blocked by binding near bridge helix. Pol III requires about ten micrograms per milliliter, intermediate sensitivity, while Pol I remains active even at hundreds of micrograms per milliliter, classified as resistant. Ranking therefore Pol II greater than Pol III greater than Pol I. Structural divergence in funnel domains and trigger loop residues that contact bicyclic peptide explains differential binding affinities and differential toxin susceptibility among polymerases.

Ref: Berg et al., Biochemistry, 9th ed., Chapter 28: Sensitivity II > III > I; Alberts et al., Molecular Biology of the Cell, Amanitin sensitivity ranking

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Transcription in Eukaryotes

α-amanitin strongly inhibits

Alpha-amanitin cyclic octapeptide from Amanita phalloides death cap mushroom penetrates hepatocytes and binds with extremely high affinity in funnel and trigger loop beneath RNA polymerase II active site, obstructing translocation and nucleotide incorporation, leading to slow elongation arrest and degradation of RPB1 subunit. RNA polymerase II is most sensitive with inhibition constant around ten nanomolar, blocked at one microgram per milliliter, causing fatal liver failure. Polymerase III requires tenfold higher concentration, while polymerase I and mitochondrial polymerase resist up to high doses due to structural differences.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10: alpha-amanitin strongly inhibits Pol II; Berg et al., Biochemistry, Amanitin mechanism

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Transcription in Eukaryotes

Phosphorylation of Ser2 in CTD is associated with

During transition to productive elongation, CDK9 cyclin-dependent kinase of P-TEFb complex and later CDK12 phosphorylate serine two of CTD heptad repeats YSPTSPS, creating high density pattern near three prime end of gene body. Serine2-P CTD recruits histone chaperone FACT, Spt6, chromatin modifier Set2 responsible for H3K36 trimethylation, and cleavage and polyadenylation factors CPSF and CstF for messenger RNA three prime end formation. Exchange from serine five to serine two ensures temporal coupling of elongation, chromatin restoration, and termination. Phosphatase Fcp1 recycles polymerase after termination releasing from DNA.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 10: Ser2-P marks elongation; J Biol Chem, CTD Ser2 phosphorylation elongation

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