Regulating gene expression Goal is controlling Proteins How many? Where? How active? 8 levels (two not shown are mRNA localization & prot degradation)

Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor 50 % of total RNA synthesis insensitive to -aminitin Mg2+ cofactor Regulated @ initiation frequency

RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA & scRNA) >100 different kinds of genes ~10% of all RNA synthesis Cofactor = Mn2+ cf Mg2+ sensitive to high [-aminitin]

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

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) Kinase CTD negative charge gets it started 3) Exchange initiation for elongation factors 4) Continues until hits terminator

Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) Kinase CTD negative charge gets it started 3) RNA pol II is paused on many promoters! even of genes that aren’t expressed! 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

NTP enters E site & rotates into A site Transcription NTP enters E site & rotates into A site

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

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

Specificity comes from trigger loop 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 http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001715 lncRNA displaces glucose-responsive repressors & co- repressors from genes for galactose catabolism

Activated transcription by Pol II Other lncRNA “promote transcriptional poising” in yeast http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001715 lncRNA displaces glucose-responsive repressors & co- repressors from genes for galactose catabolism Speeds induction of GAL genes

Euk gene regulation Initiating transcription is 1st & 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

Epigenetics heritable chromatin modifications are associated with activated & repressed genes

Epigenetics ChIP-chip & ChiP-seq data for whole genomes yield complex picture: 17 mods are associated with active genes in CD-4 T cells

Generating methylated DNA Si RNA are key: generated from antisense or foldbackRNA

Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3

Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA

Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA RDR2 makes bottom strand

Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA RDR2 makes bottom strand DCL3 cuts dsRNA into 24nt 2˚ siRNA

Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA RDR2 makes bottom strand DCL3 cuts dsRNA into 24nt 2˚ siRNA Amplifies signal!-> extends Methylated region

Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA RDR2 makes bottom strand DCL3 cuts dsRNA into 24nt 2˚ siRNA Amplifies signal!-> extends Methylated region These guide “silencing Complex” to target site (includes Cytosine & H3K9 Methyltransferases)

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 1) Capping 2) Splicing: removal of introns Evidence: electron microscopy sequence alignment

Splicing: the spliceosome cycle 1) U1 snRNP (RNA/protein complex) binds 5’ splice site

Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint

Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint 3) U4/U5/U6 complex binds intron displace U1 spliceosome has now assembled

Splicing: RNA is cut at 5’ splice site cut end is trans-esterified to branchpoint A

Splicing: 5) RNA is cut at 3’ splice site 6) 5’ end of exon 2 is ligated to 3’ end of exon 1 7) everything disassembles -> “lariat intron” is degraded

Splicing:The spliceosome cycle

Splicing: Some RNAs can self-splice! role of snRNPs is to increase rate! Why splice?

Splicing: Why splice? 1) Generate diversity exons often encode protein domains

Splicing: Why splice? 1) Generate diversity exons often encode protein domains Introns = larger target for insertions, recombination

Why splice? 1) Generate diversity >94% of human genes show alternate splicing

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant stress-response proteins

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant Stress-response proteins Splice-regulator proteins control AS: regulated by cell-specific expression and phosphorylation

Splicing: Why splice? 1) Generate diversity 2) Modulate gene expression introns affect amount of mRNA produced

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 codon @ 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 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 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 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: 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 >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 10-35 b 3’ to AAUAAA

mRNA Processing: Polyadenylation 3) PAP (PolyA polymerase) binds & cleaves 10-35 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 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!