Activated transcription by Pol IIenhancers are sequences 5’ to TATAA

transcriptional activators bind them• have distinct DNA binding and activation domains

Activated transcription by Pol IIenhancers 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 IIenhancers 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 yeasthttp://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001715•lncRNA displacesglucose-responsiverepressors & co-repressors from genesfor galactose catabolism•Speeds induction ofGAL genes

Euk gene regulationInitiating transcription is 1st & most important controlMost genes are condensedonly express needed genesnot enough room in nucleus toaccess 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 PROCESSINGPrimary transcript is hnRNAundergoes 3 processing reactions before export to cytosolAll three are coordinated with transcription & affect gene expression: enzymes piggy-back on POLII

mRNA PROCESSINGPrimary transcript is hnRNAundergoes 3 processing reactions before export to cytosol1) Capping addition of 7-methyl G to 5’ end

mRNA PROCESSINGPrimary transcript is hnRNAundergoes 3 processing reactions before export to cytosol1) Capping addition of 7-methyl G to 5’ end

identifies it as mRNA: needed for export & translation

mRNA PROCESSINGPrimary transcript is hnRNAundergoes 3 processing reactions before export to cytosol1) Capping addition of 7-methyl G to 5’ end

identifies it as mRNA: needed for export & translationCatalyzed 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 editingHuman intestines edit APOB mRNA C -> U to create a stop codon @ aa 2153 (APOB48) cf full-length APOB100• APOB48 lacks the CTD LDL receptor binding site

mRNA Processing: RNA editingHuman intestines edit APOB mRNA C -> U to create a stop codon @ 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 editingTwo 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 editingTwo 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 200- 250 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: PolyadenylationAddition of 200- 250 As to end of mRNAWhy 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: Polyadenylationsome thalassemias are due to mis-poly AInfluenza 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: Polyadenylation1) CPSF binds AAUAAA in hnRNA2) CStF (Cleavage Stimulatory Factor) binds G/U rich sequence 50 bases downstream

CFI, CFII bind in between

Polyadenylation1) CPSF binds AAUAAA in hnRNA2) CStF binds; CFI, CFII bind in between3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA

mRNA Processing: Polyadenylation3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA4) PAP adds As slowly, CFI, CFII and CPSF fall off

mRNA Processing: Polyadenylation4) PAP adds As slowly, CFI, CFII and CPSF fall off5) PABII binds, add As rapidly until 250

Coordination of mRNA processingSplicing 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 NucleusOccurs through nuclear poresanything > 40 kDa needs exportinproteinbound 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 regulation1) mRNA processing2) export from nucleus3) mRNA degradation 4) mRNA localization• RNA-binding proteinslink it to cytoskeleton:bring it to correct siteor 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 theplant 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 todestroy PHO2 mRNA upon Pi stress•PHO2 negatively regulates Pi uptake

Post-transcriptional regulationRNA in pollen controls first division after fertilization!

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

Post-transcriptional regulation4) 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 regulation4) 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 Prokaryotes1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA

Initiation in Prokaryotes1) 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 Prokaryotes1) 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 Prokaryotes1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2) Complex scans down until finds Shine-Dalgarno sequence, 16S

rRNA binds S-D3) IF2-GTP binds tRNAi

fMet

complex binds start codon

Initiation in Prokaryotes1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA 2) Complex scans down until finds Shine-Dalgarno sequence, 16S

rRNA binds S-D3) IF2-GTP binds tRNAi

fMet

complex binds start codon4) Large subunit binds

IF2-GTP -> IF2-GDPtRNAi

fMet is in P siteIFs fall off

Elongation1) EF-Tu brings charged tRNA into A site

Elongation1) EF-Tu brings charged tRNA into A site• anticodon bindsmRNA codon, EF-Tu-GTP -> EF-Tu-GDP

Elongation1) EF-Tu brings charged tRNA into A site• anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA at A site

Elongation1) EF-Tu brings charged tRNA into A site• anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA at A site peptidyl transferase is 23S rRNA!

Elongation3) 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

Wobbling1st base of anticodon can form unusual pairs with 3rd base of codon

Wobbling1st base of anticodon can form unusual pairs with 3rd base of codon

Wobbling1st base of anticodon can form unusual pairs with 3rd base of codon•Reduces # tRNAs needed: • bacteria have 40 or less (bare minimum is 31 + initiator tRNA)• Eukaryotes have ~ 50

Termination1) Process repeats until a stop codon is exposed2) release factor binds nonsense codon •3 stop codons = 3 RF in prokaryotes (1 RF binds all 3 stop codons in euk)

Termination1) Process repeats until a stop codon is exposed2) 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 site4) Ribosome falls apart