-The Prokaryotic Lac Operon is classic model of DNA/protein regulation

1. Enzyme production in prokaryotes (bacteria) is a model for how DNA is converted to protein (enzymes) in eukaryotic cells December 3 and 5 -The Prokaryotic Lac Operon is classic model of DNA/protein regulation • Repressor binding/unbinding is key to mRNA production • How is protein creation similar and different in prokaryotes and eukaryotes? -Eukaryotic Expression of Genes: -What changes occur in the genes themselves? -Modification of mRNA production rate: 3 types of transcription factor: Initiators, Promotors/Suppressors, Enhancers/Silencers -What methods are used by transcription factors to bind mRNA? -How do Hormone Responsive Elements modify transcription? -How is protein production modified by post-transcriptional modification of mRNA? -How do cells modify the protein turn-over rate?

2. Basics: How do bacterial cells ensure that enzymes are only produced (this is expensive) when the enzymes are needed? • Bacteria are simple models for eukaryotes- • Gene(DNA) --> Protein(enzyme) • Constituitive Gene--> enzyme made at same rate all the time • Regulated Gene--->enzyme only produced when needed • Substrate Induction: Inducible Enzyme Synthesis • End Product Repression: Inhibition of Enzyme Synthesis • Bacterial genes with similar function are organized on the DNA sequence in clusters called “Operons”

3. The Lac-Operon represents the classic model of how bacteria respond to the enzyme requirements of their environment. • 1961 Menod and Jacobs • Trick: A Regulatory Protein that binds a specific DNA sequence is produced from regulatory gene • Function Allosterically regulated when the effector molecule binds special DNA site • Lac Operon: What genes are in it? • A set of 3 genes needed for bacterial lactose metabolism! • B-galactosidase: Cuts lactose in bacterial cell • Galactoside Permease: Lets lactose into bacterial cell • Transacetylase: Attachs acetyl group to potential toxic metabolites • Promotor Site: mRNA Polymerase must bind operon DNA here! • Operator Site: DNA for regulator binding upstream of genes • Allosteric Regulation of ‘regulator’ determines affinity for DNA

4. Regulator binding to the operator sequence of DNA prevents RNA Polymerase from binding the DNA promotor sequence and thereby prevents it from making mRNA from the DNA. When effector (lactose catabolite) binds the regulator it is removed from DNA making mRNA production possible.

5. Eukaryotic and Prokaryotic cells exhibit similarities and differences with respect to how they regulate protein production from DNA. • Prokaryote vs. Eukaryote • Relative Genome Size: • How much DNA/How many genes? • Genome Complexity: • How complex is gene regulation? • Internal Compartmentalization: • Organelles? • DNA folding/structure Organization: • VIP eukaryotes have no operons • Relative mRNA Half-Life: • Post-Translational Modification: • Protein Turn-Over Rate:

6. Basics of protein regulation in a eukaryotic cell.

7. What tools are available to eukaryotic cells for regulating protein (enzyme) production? • 1) Genomics: modification of DNA make-up • 2) Transcription: modification of mRNA production • 3) RNA Processing: modify mRNA before it gets to the ribosome for translation • 4) Translation: modify way mRNA is converted to protein or modify mRNA turn-over rate • 5) Post-translation: modify protein half-life

8. What changes occur at the genome level to modify protein production rates? 1)Gene Amplification: make more copies of the same gene…makes more mRNA…makes more protein…. • Malaria resistance to drugs is a classic example! • Gene deletion can also occur…”selection pressure”…. 2)Gene Rearrangement: important for antibodies! • Splice out sections of unwanted DNA 3)Controlled DNA (chromatin) unwinding • Unwinding needed to let RNA polymerase get to DNA • Chromosomal puffs are classic example 4)DNA Base Methylation (-CH3) • Turns genes OFF in current and subsequent generations • Epigenetics https://www.youtube.com/watch?v=Tj_6DcUTRnM

9. DNA Can be modified!Genes can be spliced/ rearranged for a variety of combinations required for different mRNAs and different antibodies!EmbryonicCellAdultLymophocyteEmbryonic DNAAdultDNASomatic Cells ONLY!In the end an Ab gets a single V,D,J and C segment.Acquired immunity occurs when adult cell replicates itself and remaining gene segment combination.

10. DNA Binding Transcription factors (general and specific) are central to modifying transcription rate. • General or non-specific TF bind DNA at what is called the TATA-box and initiation site. When they bind, they attract RNA polymerase II! • Promotors are specific TF that bind close to the initiation site • Classic DNA Target sequences (TFs): GC-box, CAAT-box • Repressors can inhibit RNApolymerase binding • TFs are proteins produced by separate genes/mRNAs • Enhancers/Silencers are specific TFs binding specific DNA sequences far from gene initiation site • Up to 70,000 bases distant! • DNA strand twists back so TF contacts DNA at two locations • Location1:TF binding site Location2:Gene initiation site

11. What do promotors and enhancers look like?Suppressors/Enhancers can even be DownStream!

12. Fine tuning of transcription is provided by the regulatory transcription factors. Cells in different tissues have different regulatory factors! Therefore different tissues express their genes differently!

13. Hormone Response Elements (HREs) are special TFs that are activated when a hormone binds to them!HRE-hormone complex modifies Transcription. Four parts to an HRE: • Variable region of protein • DNA binding domain of protein • Hinge region of protein • Hormone binding domain of protein Hormones using HREs: are typically fat soluble- Vit D, Vit A, Testosterone, Estrogen, Thyroxine, Cortisol (glucocorticoids) • A carrier is typically required to let these non-water soluble hormones get to the DNA • HREs usually work via a zinc finger motif (Zn+2 straddled between 4 cysteines (-SH) • HREs can have both enhancer AND silencer activities in the same cell at the same time ON DIFFERENT GENES!

14. The glucocorticoid receptor is bound by the hormone cortisol. It promotes suppression of inflammation/growth, increased glycogen formation, decreased fat synthesis and other activities related to stress management. Cortisol can BOTH promote and suppress gene transcription at the same time in the same cell.

15. Other Tricks for modifying Transcription Factor activity. • TF Phosphorylation/Dephosphorylation • Classic: Protein kinases • TF activity is dependent on presence/absence of phosphorylation • AdenylCyclase(cAMP) and Tyrosine kinases • Cancer often results from a breakdown in second messenger degradation of improper phosphorylation • Heat Shock Proteins: TF activity/production is temperature dependent • Protein activity is temperature dependent • Allows cells to respond to changes in temperature • Homeotic TFs: control of embryonic development • Classic: Frogs and vitamin A receptors • Activation is often a sequential matter • Humans: Folate, Spina Bifida, and cell migration to generate the closure of your neural tube

16. Genes can also be regulated by events that occur AFTER the mRNA is produced. • Alternative mRNA Splicing in nucleus prior to use • Applications: Immunoglobins, N-CAMS, Fibronectin • Also important in viral evolution/drug resistance • Addition of a poly-AAAAAAtail to 3’ end after mRNA is transcribed from DNA • The length of the tail determines the mRNA half-life • Longer tail gives longer half-life • Initiation Factors can also help initiate (or repress) the affinity that ribosomes have for mRNA

17. Iron metabolism is one such example of where this process is used in a cell to promote the creation of transferrin when more iron is needed in cell.

18. Alternative mRNA splicing in the nucleus can be used to make a variety of potential immunoglobin combinations after different exons/introns are spliced out of mRNA transcript.

19. How is insulin is created inside the Beta-cells of the pancreas as a result of enhancers, promotors and RNA Polymerase? Then preproinsulin mRNA moves to cytosol, finds a ribosome, and translation (tRNAs) can occur until theSignalRecognitionPeptide is exposed. In the cytosol the SRP causes movement to rough side of ER and translocation of nascent peptide into the ER lumen.

20. SR peptide tail lets preproinsulin moves into the ER lumen, the signal peptide is removed, disulfides created, and the C-peptide removed as it moves to the Golgi Apparatus. Insulin now forms dense granules at trans face of Golgi and remains in a secretory vesicle until your blood sugar gets too high (remember how the glucose permease lets glucose enter the cell?). A Beta cell Ca++ influx that leads to vesicular secretion so insulin can enter your blood and let insulin-sensitive cells become permeable to glucose as a result of its tyrosine kinase activity. Insulin creates dark Granules in this Beta-cell.

21. Cells can also modify the turn-over rate of the proteins produced from mRNA by attaching a small 76 amino acid peptide called “ubiquitin” that signal rapid degradation in a proteosome.

22. The “other” two major types of RNA have a powerful ability to modify access to the genetic material in a gene:Small Interference RNA(siRNA) and MicroRNA(miRNA)Nobel Prize 2006: (VERY NEW!) siRNA: -20-25 nucleotides long and non-protein-coding RNA molecules that are encoded by their own genes -“Dicer” enzyme insert siRNA into an mRNA making it abnormal and causing degradation of mRNA -mRNA is effectively removed (gene is silenced) miRNA: -21-22 nucleotides long and made from fragments of GC-loop and DS RNA -Promote rapid mRNA degradation (gene is silenced)

23. siRNA and miRNA both potentially silence genes and prevent transcription of mRNA for better or worse. (Ahmadzada et al 2018) https://www.nobelprize.org/prizes/medicine/2006/advanced-information/

24. Differences between miRNA and siRNA https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803180/pdf/12551_2017_Article_392.pdf