I. Overview of Eukaryotic gene regulation

1. I. Overview of Eukaryotic gene regulation • Mechanisms similar to those found in bacteria-most genes controlled at the transcriptional level • Much more complex than prokaryotic • chromatin • TFs • Enhancers • Activators

2. A. Prokaryotes vs. Eukaryotes • In eukaryotes, one mRNA = one protein. (in bacteria, one mRNA can be polycistronic, or code for several proteins). • DNA in eukaryotes forms a stable, compacted complex with histones. In bacteria, the chromatin is not in a permanently condensed state. • Eukaryotic DNA contains large regions of repetitiveDNA, whilst bacterial DNA rarely contains any "extra" DNA. • Eukaryotic genes are divided into exons and introns; in bacteria, genes are almost never divided. • In eukaryotes, mRNA is synthesized in the nucleus and then processed and exported to the cytoplasm; in bacteria, transcription and translation can take place simultaneously off the same piece of DNA.

3. B. Eukaryote gene expression is regulated at 6 levels: Transcription RNA processing mRNA transport mRNA translation mRNA degradation Protein degradation

4. II. Transcriptional Control • Control factors • cis-acting “next to” elements • Promoter region: TATA box (-30), CAAT box (-80) GC box (-110) • Alternate promoters • The level of transcription initiation can vary between alternative promoters • the translation efficiency of mRNA isoforms with different leader exons can differ • alternative promoters can have different tissue specificity and react differently to some signals • Enhancers & Silencers far away from promoter • trans-acting “across from” factors • Transcription factors • Activators, Coactivators

5. Control factors continued: • DNA methylation (add methyl to C) • Occurs at 5’ position, usually in CG doublets • 5’-mCpG-3’ • Inverse relationship between degree of methylation and degree of expression • Not 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.

7. Control factors continued: • Chromatin conformation (remodelling) • Antirepressors & nucleosome positioning. • Histone acetylation – (acetyl groups on lysines), histone acetyltransferase enzyme catalyzes the addition of lysine, targeted to genes by specific TFs. • Heterochromatin – highly condensed, transcriptionally inert (off).

10. B. Eukaryotic Promoters Usually located within 100 bp upstream Usually contains TATA box (25 – 30) bases upstream from start point, additional elements: • CAAT box • GC box • Recognized byRNA Pol II (transcribes mRNA) • Require the binding of several protein factors to initiate transcription (DNA binding domains on TFs – ‘motifs’) • May be positively or negatively regulated

11. C. Transcription Factors –the transcription complex • TFIIA, TFIIB, TFIID, TFIIE, TFIIH • TATA binding protein (TBP) • TBP associated factors (TAFs)

12. 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

13. Enhancers Cis 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.

14. D. An example of transcriptional control: Galactose metabolism in yeast • GAL1, GAL7, GAL10 genes… products required for conversion of galactose into glucose Closely linked genes, but monocistronic mRNAs synthesized These are only transcribed when galactose is present…

15. Galactose metabolizing pathway of yeast.

16. Controlling GAL GAL80 encodes a protein that negatively regulates transcription. The repressor protein binds to an Activator protein, rendering it inactive. GAL4 encodes an activator w/zinc finger motif that activates transcription of the three GAL genes individually. Galactose = Inducer, that binds to Gal80, causing it to release Gal4 • Although this looks similar to Lac Operon, there are different molecular mechanisms…

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

18. Activation model of GAL genes in yeast.

19. III. Post-transcriptional control • Alternative splicing - Some messages undergo alternate splicing depending on what tissue they are located in. The regulation is at the level of snRNP production. • Some pre-mRNAs can be spliced in more than one way, producing 2+ alternative mRNA’s • Can introduce stop codons or change the reading frame • Controlled by RNA binding splicing factors that commit splicing in a particular way

20. Alternative polyadenylation and splicing of the human CACL gene in thyroid and neuronal cells. Calcitonin gene-related peptide

21. Post-transcriptional control cont. • The stability of a class of mRNA can be controlled. • Some short-lived mRNAs have multiple copies of the sequence AUUUA which may act as a target for degradation. • the hormone prolactin enhances the stability of the mRNA for the milk protein casein • high levels of iron decrease the stability of the mRNA for the receptor that brings iron into cells • RNA interference – poorly understood, but appears to be widespread in fungi, plants and animals as a regulatory mechanism • miRNAs & siRNAS (small RNA molecules) pair with proteins to form an RNA-induced silencing complex (RISC) • RISC pairs w/complentary base sequences of specific mRNAs and causes: • Cleavage of mRNA • Inhibition of translation • Transcriptional silencing • Degradation of mRNA