Regulating gene expressionGoal is controlling Proteins•How many?•Where?•How active?8 levels (two notshown are mRNAlocalization & protdegradation)

Transcription in EukaryotesPol 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 synthesisCofactor = 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 IIBasal transcription1) Once assemble initiation complex must start Pol II2) Kinase CTD

negative charge gets it started

3) Exchange initiation for elongation factors4) Continues untilhits terminator

Initiation of transcription by Pol IIBasal transcription1) Once assemble initiation complex must start Pol II2) Kinase CTD

negative charge gets it started

3) RNA pol II is pausedon many promoters!• even of genes thataren’t expressed!•Early elongation is also regulated!

Initiation of transcription by Pol IIRNA 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 IIRNA 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 IIRNA 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!

TranscriptionTemplate strand determines next basePositioned by H-bondsuntil RNA polymeraselinks 5’ P to 3’ OH in front

TranscriptionTemplate strand determines next basePositioned by H-bondsuntil RNA polymeraselinks 5’ P to 3’ OH in frontEnergy comes from hydrolysisof 2 Pi

TranscriptionNTP enters E site & rotates into A site

TranscriptionNTP enters E site & rotates into A siteSpecificity comes from trigger loop

TranscriptionSpecificity comes from trigger loopMobile motif that swings into position & triggers catalysis

TranscriptionSpecificity comes from trigger loopMobile motif that swings into position & triggers catalysisRelease of PPi triggers translocation

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

Activated transcription by Pol IIStudied by mutating promoters for reporter genes

Activated transcription by Pol IIStudied by mutating promoters for reporter genesRequires transcription factors and changes in chromatin

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

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

Epigenetics•heritable chromatin modifications are associated with activated & repressed genes

EpigeneticsChIP-chip & ChiP-seq data for whole genomes yieldcomplex picture: 17 mods are associated with active genes in CD-4 T cells

Generating methylated DNASi RNA are key: generated from antisense or foldbackRNA

Generating methylated DNASi RNA are from antisense or foldback RNAPrimary 24 nt siRNA are generated by DCL3

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

Generating methylated DNASi RNA are from antisense or foldback RNAPrimary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNARDR2 makes bottom strand

Generating methylated DNASi RNA are from antisense or foldback RNAPrimary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNARDR2 makes bottom strandDCL3 cuts dsRNA into 24nt2˚ siRNA

Generating methylated DNASi RNA are from antisense or foldback RNAPrimary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNARDR2 makes bottom strandDCL3 cuts dsRNA into 24nt2˚ siRNAAmplifies signal!-> extendsMethylated region

Generating methylated DNASi RNA are from antisense or foldback RNAPrimary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNARDR2 makes bottom strandDCL3 cuts dsRNA into 24nt2˚ siRNAAmplifies signal!-> extendsMethylated regionThese guide “silencingComplex” to target site(includes Cytosine & H3K9 Methyltransferases)

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 PROCESSING1) Capping2) Splicing: removal of intronsEvidence:• electron microscopy• sequence alignment

Splicing: the spliceosome cycle

1) U1 snRNP (RNA/protein complex) binds 5’ splice site

Splicing:The spliceosome cycle1) U1 snRNP binds 5’ splice site2) U2 snRNP binds “branchpoint”

-> displaces A at branchpoint

Splicing:The spliceosome cycle1) U1 snRNP binds 5’ splice site2) U2 snRNP binds “branchpoint”

-> displaces A at branchpoint3) U4/U5/U6 complex binds intron

displace U1spliceosome has now assembled

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

Splicing:5) RNA is cut at 3’ splice site6) 5’ end of exon 2 is ligated to 3’ end of exon 17) 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 diversityexons often encode protein domainsIntrons = 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 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!