I. Overview of Eukaryotic gene I. Overview of Eukaryotic gene regulationregulation

Mechanisms similar to Mechanisms similar to those found in bacteria-those found in bacteria-most genes controlled most genes controlled at the transcriptional at the transcriptional levellevelMuch more complex Much more complex than prokaryoticthan prokaryotic

chromatinchromatin TFsTFs EnhancersEnhancers ActivatorsActivators

A. Prokaryotes vs. EukaryotesA. Prokaryotes vs. Eukaryotes In eukaryotes, In eukaryotes, one mRNA = one proteinone mRNA = one protein. .

(in bacteria, one mRNA can be polycistronic, or code (in bacteria, one mRNA can be polycistronic, or code for several proteins). for several proteins).

DNA in eukaryotes forms a stable, compacted complex DNA in eukaryotes forms a stable, compacted complex with with histoneshistones. In bacteria, the chromatin is not in a . In bacteria, the chromatin is not in a permanently condensed state. permanently condensed state.

Eukaryotic DNA contains large regions of Eukaryotic DNA contains large regions of repetitiverepetitive DNADNA, whilst bacterial DNA rarely contains any "extra" , whilst bacterial DNA rarely contains any "extra" DNA. DNA.

Eukaryotic genes are divided into Eukaryotic genes are divided into exons and intronsexons and introns; in ; in bacteria, genes are almost never divided. bacteria, genes are almost never divided.

In eukaryotes, mRNA is In eukaryotes, mRNA is synthesized in the nucleussynthesized in the nucleus and and then processed and exported to the cytoplasm; in then processed and exported to the cytoplasm; in bacteria, transcription and translation can take place bacteria, transcription and translation can take place simultaneously off the same piece of DNA. simultaneously off the same piece of DNA.

B. Eukaryote gene expression is regulated at 6 levels:

1. Transcription

2. RNA processing

3. mRNA transport

4. mRNA translation

5. mRNA degradation

6. Protein degradation

II. Transcriptional ControlII. Transcriptional ControlA.A. Control factorsControl factors

1)1) ciscis-acting “next to” elements-acting “next to” elementsPromoter region: TATA box (-30), CAAT box Promoter region: TATA box (-30), CAAT box (-80) GC box (-110)(-80) GC box (-110)Alternate promotersAlternate promoters The level of transcription initiation can vary between The level of transcription initiation can vary between

alternative promoters alternative promoters the translation efficiency of mRNA isoforms with the translation efficiency of mRNA isoforms with

different leader exons can differdifferent leader exons can differ alternative promoters can have different tissue alternative promoters can have different tissue

specificity and react differently to some signalsspecificity and react differently to some signalsEnhancers & Silencers far away from promoterEnhancers & Silencers far away from promoter

2)2) transtrans-acting “across from” factors-acting “across from” factorsTranscription factorsTranscription factorsActivators, CoactivatorsActivators, Coactivators

Control factors continued:Control factors continued:3)3) DNA methylation (DNA methylation (add methyl to Cadd methyl to C))

Occurs at 5’ position, usually in CG doubletsOccurs at 5’ position, usually in CG doublets 5’-5’-mmCpG-3’CpG-3’ Inverse relationship between degree of methylation Inverse relationship between degree of methylation

and degree of expressionand degree of expression Not a general mechanism in eukaryotesNot a general mechanism in eukaryotes

Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes.

A gene for methylation is essential for development in mice (turning off a gene also can be important).

Methylation results in a human disease called fragile X syndrome; FMR-1 gene is silenced by methylation.

Control factors continued:Control factors continued:

4)4) Chromatin conformation (remodelling)Chromatin conformation (remodelling)a.a. Antirepressors & nucleosome positioning.Antirepressors & nucleosome positioning.

b.b. Histone acetylation – (acetyl groups on lysines), Histone acetylation – (acetyl groups on lysines), hhistone istone aacetylcetylttransferase enzyme catalyzes the ransferase enzyme catalyzes the addition of lysine, targeted to genes by specific addition of lysine, targeted to genes by specific TFs.TFs.

c.c. Heterochromatin – highly condensed, Heterochromatin – highly condensed, transcriptionally inert (off).transcriptionally inert (off).

B. Eukaryotic PromotersB. Eukaryotic PromotersUsually located within 100 bp upstreamUsually located within 100 bp upstreamUsually contains Usually contains TATA boxTATA box (25 – 30) bases upstream from start point, additional (25 – 30) bases upstream from start point, additional

elements:elements: CAAT boxCAAT box GC boxGC box

Recognized byRNA Pol II (transcribes mRNA)Recognized byRNA Pol II (transcribes mRNA) Require the binding of several protein factors to initiate transcription (DNA Require the binding of several protein factors to initiate transcription (DNA

binding domains on TFs – ‘motifs’)binding domains on TFs – ‘motifs’) May be positively or negatively regulatedMay be positively or negatively regulated

C. Transcription Factors –the C. Transcription Factors –the transcription complextranscription complex

1)1) TFIIA, TFIIB, TFIIA, TFIIB, TFIID, TFIIE, TFIID, TFIIE, TFIIHTFIIH

2)2) TATA binding TATA binding protein (TBP)protein (TBP)

3)3) TBP associated TBP associated factors (TAFs)factors (TAFs)

Assembly of the basal transcription apparatus - involves stepwise binding of various transcription factor proteins.

These trans-acting proteins are required for RNA pol II to initiate transcription.

Commitment Stage & Clearance Stage…

Activators are required to bring about normal levels of transcription

EnhancersCis regulators that interact with regulatory proteins & TFs to increase the efficiency of transcription initiation.

Silencers – cis-acting, bound by repressors, or cause the chromatin to condense and become inactive.

Activators - Proteins that function by contacting basal transcription factors and stimulating the assembly of pre-initiation complexes at promoters.

D. An example of transcriptional control: D. An example of transcriptional control: Galactose metabolism in yeastGalactose metabolism in yeast

GAL1, GAL7, GAL10GAL1, GAL7, GAL10 genes… products genes… products required for conversion of galactose into required for conversion of galactose into glucoseglucose

Closely linked genes, but monocistronic mRNAs synthesized These are only transcribed when galactose is present…

Galactose metabolizing pathway of yeast.

Controlling Controlling GALGAL

GAL80GAL80 encodes a protein that negatively regulates encodes a protein that negatively regulates transcription. The repressor protein binds to an transcription. The repressor protein binds to an Activator protein, rendering it inactive.Activator protein, rendering it inactive.

GAL4GAL4 encodes an activator w/zinc finger motif that encodes an activator w/zinc finger motif that activates transcription of the three activates transcription of the three GALGAL genes genes individually.individually.

Galactose = Inducer, that binds to Gal80, causing Galactose = Inducer, that binds to Gal80, causing it to release Gal4it to release Gal4

Although this looks similar to Lac Operon, there Although this looks similar to Lac Operon, there are different molecular mechanisms…are different molecular mechanisms…

Two trans-acting genes (GAL4 and GAL80) and one upstream cis-acting locus (UAS) work to regulate galactokinase synthesis.

Activation model of GAL genes in yeast.

III. Post-transcriptional controlIII. Post-transcriptional control

A.A. Alternative splicing - Some messages undergo Alternative splicing - Some messages undergo alternate splicingalternate splicing depending on what tissue they depending on what tissue they are located in. The regulation is at the level of are located in. The regulation is at the level of snRNP production. snRNP production.

Some pre-mRNAs can be Some pre-mRNAs can be spliced in more than one wayspliced in more than one way, , producing 2+ alternative mRNA’sproducing 2+ alternative mRNA’s

Can introduce stop codons or change the reading frameCan introduce stop codons or change the reading frame Controlled by RNA binding splicing factors that commit Controlled by RNA binding splicing factors that commit

splicing in a particular waysplicing in a particular way

Alternative polyadenylation and splicing of the human CACL gene in thyroid and neuronal cells.

Calcitonin gene-related peptide

Post-transcriptional control cont.Post-transcriptional control cont.B.B. The stability of a class of mRNA can be controlled. The stability of a class of mRNA can be controlled.

Some short-lived mRNAs have multiple copies of the sequence Some short-lived mRNAs have multiple copies of the sequence AUUUA which may act as a target for degradation. AUUUA which may act as a target for degradation.

the hormone prolactin enhances the stability of the mRNA for the the hormone prolactin enhances the stability of the mRNA for the milk protein casein milk protein casein

high levels of iron decrease the stability of the mRNA for the high levels of iron decrease the stability of the mRNA for the receptor that brings iron into cells receptor that brings iron into cells

C.C. RNA interference – poorly understood, but appears to be RNA interference – poorly understood, but appears to be widespread in fungi, plants and animals as a regulatory widespread in fungi, plants and animals as a regulatory mechanismmechanism

miRNAs & siRNAS (small RNA molecules) pair with proteins to miRNAs & siRNAS (small RNA molecules) pair with proteins to form an RNA-induced silencing complex (form an RNA-induced silencing complex (RISCRISC))

RISC pairs w/complentary base sequences of specific mRNAs and RISC pairs w/complentary base sequences of specific mRNAs and causes: causes:

1)1) Cleavage of mRNACleavage of mRNA2)2) Inhibition of translationInhibition of translation3)3) Transcriptional silencingTranscriptional silencing4)4) Degradation of mRNADegradation of mRNA