Transcription in Eukaryotes 3 RNA polymerases all are multi-subunit complexes 5 in common 3 very similar variable # unique ones Now have Pols IV & V in plants Make siRNA

Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor 50 % of total RNA synthesis insensitive to  -aminitin Mg 2+ cofactor initiation frequency

RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA & scRNA) >100 different kinds of genes ~10% of all RNA synthesis Cofactor = Mn 2+ cf Mg 2+ sensitive to high [  -aminitin]

RNA Polymerase II makes mRNA (actually hnRNA), some snRNA and scRNA ~ 30,000 different genes 20-40% of all RNA synthesis very sensitive to  -aminitin

Initiation of transcription by Pol II Separate basal and activated transcription activated transcription is regulated by proteins bound to promoter elements called enhancers and silencers usually 5’ to TATAA box TATAA coding sequence UCE

Initiation of transcription by Pol II Basal transcription 1) TFIID (includingTBP) binds TATAA box

Initiation of transcription by Pol II Basal transcription 1) TFIID binds to TATAA box 2) Distorts DNA

Initiation of transcription by Pol II Basal transcription 1) TFIID binds TATAA box 2) TFIIA and TFIIB bind TFIID/DNA

Initiation of transcription by Pol II Basal transcription 1) TFIID binds TATAA box 2) TFIIA and TFIIB bind TFIID/DNA 3) Complex recruits Pol II

Initiation of transcription by Pol II Basal transcription 1) TFIID binds TATAA box 2) TFIIA and TFIIB bind to TFIID/DNA 3) Complex recruits Pol II 4) Still must recruit TFIIE & TFIIH to form initiation complex

Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) TFIIH kinases CTD

Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) TFIIH kinases CTD negative charge gets it started 3) Exchange initiation for elongation factors

Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) TFIIH kinases CTD negative charge gets it started 3) Exchange initiation for elongation factors RNA pol II is paused on many promoters!

Initiation of transcription by Pol II Basal transcription 3) Exchange initiation for elongation factors RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA])

Initiation of transcription by Pol II RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA]) Early elongation is also regulated!

Initiation of transcription by Pol II RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA]) Early elongation is also regulated! PTEFb kinases CTD to stimulate processivity & processing

Initiation of transcription by Pol II RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA]) Early elongation is also regulated! PTEFb kinases CTD to stimulate processivity & processing Many genes have short transcripts

Initiation of transcription by Pol II RNA pol II is paused on many promoters! even of genes that aren’t expressed! (low [mRNA]) Early elongation is also regulated! PTEFb kinases CTD to stimulate processivity & processing Many genes have short transcripts Yet another new level of control!

Transcription Template strand determines next base Positioned by H-bonds until RNA polymerase links 5’ P to 3’ OH in front

Transcription Template strand determines next base Positioned by H-bonds until RNA polymerase links 5’ P to 3’ OH in front Energy comes from hydrolysis of 2 Pi

Transcription NTP enters E site & rotates into A site

Transcription NTP enters E site & rotates into A site Specificity comes from trigger loop

Transcription Specificity comes from trigger loop Mobile motif that swings into position & triggers catalysis

Transcription Specificity comes from trigger loop Mobile motif that swings into position & triggers catalysis Release of PPi Triggers translocation

Transcription Proofreading: when it makes a mistake it removes ~ 5 bases & tries again

Activated transcription by Pol II Studied by mutating promoters for reporter genes

Activated transcription by Pol II Studied by mutating promoters for reporter genes Requires transcription factors and changes in chromatin

Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them have distinct DNA binding and activation domains

Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them have distinct DNA binding and activation domains activation domain interacts with mediator helps assemble initiation complex on TATAA

Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them have distinct DNA binding and activation domains activation domain interacts with mediator helps assemble initiation complex on TATAA Recently identified “activating RNA”: bind enhancers & mediator

Activated transcription by Pol II Other lncRNA “promote transcriptional poising” in yeast 71%2Fjournal.pbio lncRNA displaces glucose-responsive repressors & co- repressors from genes for galactose catabolism

Activated transcription by Pol II Other lncRNA “promote transcriptional poising” in yeast 71%2Fjournal.pbio lncRNA displaces glucose-responsive repressors & co- repressors from genes for galactose catabolism Speeds induction of GAL genes

Euk gene regulation Initiating transcription is 1 st & most important control Most genes are condensed only express needed genes not enough room in nucleus to access all genes at same time! must find & decompress gene

First “remodel” chromatin: some proteins reposition nucleosomes others acetylate histones Neutralizes +ve charge makes them release DNA role of epigenetics

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol All three are coordinated with transcription & affect gene expression: enzymes piggy-back on POLII

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end identifies it as mRNA: needed for export & translation

mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end identifies it as mRNA: needed for export & translation Catalyzed by CEC attached to POLII

mRNA Processing: RNA editing Two types: C->U and A->I

mRNA Processing: RNA editing Two types: C->U and A->I Plant mito and cp use C -> U >300 different editing events have been detected in plant mitochondria: some create start & stop codons

mRNA Processing: RNA editing Two types: C->U and A->I Plant mito and cp use C -> U >300 different editing events have been detected in plant mitochondria: some create start & stop codons: way to prevent nucleus from stealing genes!

mRNA Processing: RNA editing Human intestines edit APOB mRNA C -> U to create a stop aa 2153 (APOB48) cf full-length APOB100 APOB48 lacks the CTD LDL receptor binding site

mRNA Processing: RNA editing Human intestines edit APOB mRNA C -> U to create a stop aa 2153 (APOB48) cf full-length APOB100 APOB48 lacks the CTD LDL receptor binding site Liver makes APOB100 -> correlates with heart disease

mRNA Processing: RNA editing Two types: C->U and A->I Adenosine de-aminases (ADA) are ubiquitously expressed in mammals act on dsRNA & convert A to I (read as G)

mRNA Processing: RNA editing Two types: C->U and A->I Adenosine de-aminases (ADA) are ubiquitously expressed in mammals act on dsRNA & convert A to I (read as G) misregulation of A-to-I RNA editing has been implicated in epilepsy, amyotrophic lateral sclerosis & depression

mRNA Processing: Polyadenylation Addition of As to end of mRNA Why bother? helps identify as mRNA required for translation way to measure age of mRNA ->mRNA s with < 200 As have short half-life

mRNA Processing: Polyadenylation Addition of As to end of mRNA Why bother? helps identify as mRNA required for translation way to measure age of mRNA ->mRNA s with < 200 As have short half-life >50% of human mRNAs have alternative polyA sites!

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites!

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! result : different mRNA, can result in altered export, stability or different proteins

mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! result : different mRNA, can result in altered export, stability or different proteins some thalassemias are due to mis-poly A

mRNA Processing: Polyadenylation some thalassemias are due to mis-poly A Influenza shuts down nuclear genes by preventing poly- Adenylation (viral protein binds CPSF)

mRNA Processing: Polyadenylation 1) CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA in hnRNA

mRNA Processing: Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF (Cleavage Stimulatory Factor) binds G/U rich sequence 50 bases downstream CFI, CFII bind in between

Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF binds; CFI, CFII bind in between 3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA

mRNA Processing: Polyadenylation 3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA 4) PAP adds As slowly, CFI, CFII and CPSF fall off

mRNA Processing: Polyadenylation 4) PAP adds As slowly, CFI, CFII and CPSF fall off 5)PABII binds, add As rapidly until 250

Coordination of mRNA processing Splicing and polyadenylation factors bind CTD of RNA Pol II-> mechanism to coordinate the three processes Capping, Splicing and Polyadenylation all start before transcription is done!

Export from Nucleus Occurs through nuclear pores anything > 40 kDa needs exportin protein bound to 5’ cap

Export from Nucleus In cytoplasm nuclear proteins fall off, new proteins bind eIF4E/eIF-4F bind cap also new proteins bind polyA tail mRNA is ready to be translated!

Post-transcriptional regulation 1) mRNA processing 2) export from nucleus 3) mRNA degradation 4) mRNA localization RNA-binding proteins link it to cytoskeleton: bring it to correct site or store it

4) mRNA localization RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it Some RNA (eg Knotted) are transported into neighboring cells

4) mRNA localization RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it Some RNA are transported into neighboring cells Others are transported t/o the plant in the phloem (SUT1, KN1)

4) mRNA localization RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it Some RNA are transported into neighboring cells Others are transported t/o the plant in the phloem (SUT1, KN1) Also some siRNA & miRNA!

4) mRNA localization RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it Some RNA are transported into neighboring cells Others are transported t/o the plant in the phloem (SUT1, KN1) Also some siRNA & miRNA! siRNA mediate silencing Especially of viruses & TE

4) mRNA localization RNA-binding proteins link it to cytoskeleton:bring it to correct site or store it Some RNA are transported into neighboring cells Others are transported t/o the plant in the phloem (SUT1, KN1) Also some siRNA & miRNA! siRNA mediate silencing MiR399 moves to roots to destroy PHO2 mRNA upon Pi stress PHO2 negatively regulates Pi uptake

Post-transcriptional regulation RNA in pollen controls first division after fertilization!

Post-transcriptional regulation RNA in pollen controls first division after fertilization! Delivery by pollen ensures correct development doesn’t happen unless egg is fertilized by pollen

Post-transcriptional regulation 4) mRNA localization RNA-binding proteins link it to cytoskeleton: bring it to correct site or store it many are stored in P-bodies! More than just an RNA- destruction site

Post-transcriptional regulation 4) mRNA localization RNA-binding proteins link it to cytoskeleton: bring it to correct site or store it many are stored in P-bodies! More than just an RNA- destruction site Link with initiation of translation

Initiation in Prokaryotes 1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA

Initiation in Prokaryotes 1)IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2)Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D

Initiation in Prokaryotes 1)IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2)Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D Next AUG is Start codon, must be w/in 7-13 bases

Initiation in Prokaryotes 1)IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2)Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D 3)IF2-GTP binds tRNA i fMet complex binds start codon

Initiation in Prokaryotes 1)IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2)Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D 3)IF2-GTP binds tRNA i fMet complex binds start codon 4)Large subunit binds IF2-GTP -> IF2-GDP tRNA i fMet is in P site IFs fall off

Elongation 1) EF-Tu brings charged tRNA into A site

Elongation 1) EF-Tu brings charged tRNA into A site anticodon binds mRNA codon, EF-Tu-GTP - > EF-Tu-GDP

Elongation 1) EF-Tu brings charged tRNA into A site anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP 2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA at A site

Elongation 1) EF-Tu brings charged tRNA into A site anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP 2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA at A site peptidyl transferase is 23S rRNA!

Elongation 3) ribosome translocates one codon old tRNA moves to E site & exits new tRNA moves to P site A site is free for next tRNA energy comes from EF-G-GTP -> EF-G-GDP+ Pi

Wobbling 1 st base of anticodon can form unusual pairs with 3 rd base of codon

Wobbling 1 st base of anticodon can form unusual pairs with 3 rd base of codon

Wobbling 1 st base of anticodon can form unusual pairs with 3 rd base of codon Reduces # tRNAs needed: bacteria have 40 or less (bare minimum is 31 + initiator tRNA) Eukaryotes have ~ 50

Termination 1) Process repeats until a stop codon is exposed 2) release factor binds nonsense codon 3 stop codons = 3 RF in prokaryotes (1 RF binds all 3 stop codons in euk)

Termination 1) Process repeats until a stop codon is exposed 2) release factor binds nonsense codon 3 stop codons = 3 RF in prokaryotes (1 RF binds all 3 stop codons in euk) 3) Releases peptide from tRNA at P site 4) Ribosome falls apart

Poisons Initiation: streptomycin, kanamycin Elongation: peptidyl transferase: chloramphenicol (prok) cycloheximide (euk) translocation erythromycin (prok) diptheria toxin (euk) Puromycin causes premature termination

Initiation in Eukaryotes 1)eIF4E binds mRNA cap Won’t bind unless 5’cap is methylated