1. Medical Genetics & Genomics Guri Tzivion, PhD tzivion@windsor.edu Extension 506 BCHM 590: Fall 2015 Windsor University School of Medicine

2. Questions on Prokaryotic gene regulation?

3. Negative Regulation Example 1: The trp Operon

4. Negative Regulation Example 2: The lac Operon

5. The lac and trp operons

6. The ara operon, an example of positive transcriptional regulation When arabinose levels are high, arabinose binds to AraC, allowing it to bind to the initiator region of the ara operon. This induces the binding of the RNA polymerase to the promoter region of the ara operon, initiating the transcription of ara operon genes.

7. Negative and positive regulation of the lac operon

8. 1. Predict the phenotype of a lacI mutant: a. The lac genes would be expressed efficiently only in the absence of lactose. b. The lac genes would be expressed efficiently only in the presence of lactose. c. The lac genes would be expressed continuously. d. The lac genes would never be expressed efficiently.

9. 1. Predict the phenotype of a lacI mutant: a. The lac genes would be expressed efficiently only in the absence of lactose. b. The lac genes would be expressed efficiently only in the presence of lactose. c. The lac genes would be expressed continuously. d. The lac genes would never be expressed efficiently.

10. What would be the effect of a mutation in the lacI gene that blocks the binding of lactose to the repressor? a. The lacZYA genes would be expressed constitutively. b. The lacZYA genes would not be expressed. c. The lacZYA genes would be repressed by lactose. d. The lacZYA genes would be inducible by lactose.

11. What would be the effect of a mutation in the lacI gene that blocks the binding of lactose to the repressor? a. The lacZYA genes would be expressed constitutively. b. The lacZYA genes would not be expressed. c. The lacZYA genes would be repressed by lactose. d. The lacZYA genes would be inducible by lactose.

12. In the tryptophan operon, tryptophan serves as ________________. a) a corepressor b) an inducer c) a catalyst d) a regulatory protein

13. In the tryptophan operon, tryptophan serves as ________________. a) a corepressor b) an inducer c) a catalyst d) a regulatory protein

14. A cell does not want to waste energy synthesizing the amino acid arginine if arginine is present in its diet. Therefore, when arginine is present, it activates a repressor protein to prevent transcription of genes that code for enzymes that make arginine. This is an example of what type of regulation? a) positive b) negative-repressible c) negative-inducible d) attenuation

15. A cell does not want to waste energy synthesizing the amino acid arginine if arginine is present in its diet. Therefore, when arginine is present, it activates a repressor protein to prevent transcription of genes that code for enzymes that make arginine. This is an example of what type of regulation? a) positive b) negative-repressible c) negative-inducible d) attenuation

16. BCHM 590 MD2 Genetics Class 1 DNA: Structure, Replication and Regulation of Gene Expression 5. Regulation of gene expression: Eukaryotes

17. Regulation of gene expression in eukaryotes

18. Regulation of Gene Expression Specificity of function is determined by regulation of gene expression Gene products will be produced only ‘when & where’ they are needed and at specific levels Specific transcription factors can determine the ‘when and where’ by controlling the rate of gene transcription, for example by affecting the binding of the RNA polymerase to DNA There are nearly 2000 proteins with DNA binding domains in the genome (10% of the protein coding transcripts)

20. AP Biology Points of control The control of gene expression can occur at any step in the pathway from gene to functional protein 1.Packing/unpacking of DNA 2.Transcription 3.mRNA processing 4.mRNA transport 5.Translation 6.Protein processing 7.Protein degradation

21. Chromatin Structure Processes that modify chromatin structure can affect the expression of genes: Histone modifications can dictate weather a region of the chromosome will be transcriptionally active or inactive. These modifications include acetylation, phosphorylation, methylation and ubiquitination. Acetylation (addition of an acetyl - CH 3 CO group) for example, results in decreased condensation of the DNA and increased transcription of genes in that region. Acetylation (addition of an acetyl - CH 3 CO group) for example, results in decreased condensation of the DNA and increased transcription of genes in that region. Ubiquitination (addition of a ubiquitin polypeptide) of various histones also modifies the transcriptional activity.

23. Histone deacetylases (HDACs) are negative regulators of transcription Histone acetylases (HATs) are positive regulators of transcription In general… + - CBP/P300 HDAC1/2

25. DNA methylation DNA can be modified by methylation of adenine and cytosine bases Methylated BaseMethylation Sequence C5-methylcytosine (5-mC)CpG C5-hydroxymethylcytosine (5-hmC)CpG, CpHpG1, CpHpH1 H = Adenine, Cytosine, or Thymine "p" in CpG refers to the phosphodiester bond

27. Components of epigenetic inheritence

28. Epigenetics Epigenetics - regulation of gene expression that is not at the level of the DNA sequence

29. Imprinting expressed differently according to the parent of origin always either the version from the mother or father Igf2 is imprinted on the chromosome inherited from the mother H19 is imprinted on the paternal chromosome genes that escape epigenetic reprogramming

30. Transcriptional regulatory elements in eukaryotes

34. Control of transcription: promoters, enhancers, repressors…. Control of transcription in eukaryotes depends on both cis- and trans-acting factors, including: regulatory & promoter sequences, RNA polymerase, the general transcription factors and gene specific activators and repressors

35. Cis- versus trans-acting factors Cis: includes all the elements that are present on the same DNA strand as the regulated gene: promoter, enhancer, intron/exons etc. (addition of an acetyl - CH 3 CO group) for example, results in decreased condensation of the DNA and increased transcription of genes in that region. Cis: includes all the elements that are present on the same DNA strand as the regulated gene: promoter, enhancer, intron/exons etc. (addition of an acetyl - CH 3 CO group) for example, results in decreased condensation of the DNA and increased transcription of genes in that region. Trans: includes all the rest regulatory elements such as transcription activators and repressors. Trans: includes all the rest regulatory elements such as transcription activators and repressors.

36. Pol II requires assembly of the general TFs on the promoter in order to initiate transcription specific gene transcription may also be regulated by activator and repressor proteins binding at near OR distant sites RNA Pol II promoters vary significantly in structure but often include one or more of: TATA, GC or CAAT box However none are essential and many promoters lack them all!!

38. Classes of Transcription Factors All have at least two functional domains or regions: Transactivation domain Transactivation domain interacts with other proteins DNA-binding domain DNA-binding domain binds a specific DNA sequence several common structural protein motifs that TF use: Zinc finger Helix-turn-helix Leucine zipper Helix-loop-helix

40. Nuclear receptor transcription factors: ERalpha, AR Nuclear/steroid receptors have a steroid-binding domain required for efficient DNA binding and activation of transcription

42. Transcriptional repressors can function by competing with transcriptional activators

46. Transcript variation one gene = many mRNAs At least half of all genes have 2 or more promoters

47. Transcript variation allows multiple protein isoforms

48. Regulation of mRNA stability & degradation The stability of mRNAs is very variable A deadenylase enzyme acts to shorten the poly-A tail in the cytoplasm Decapping enzymes - uncapped mRNA is rapidly degraded by exonucleases RNAi mediated degradation

53. Control at the protein level Regulation of protein folding, often with the help of molecular chaperones (such as Hsp70) The ubiquitin-proteasome system efficiently destroys incorrectly or incompletely folded proteins and acts to limit the life of normal or correctly folded proteins Abnormally folded proteins can form disease-causing protein aggregates (e.g., neurodegenerative diseases) Regulation of protein function: phosphorylation, acetylation and protein-protein interactions

54. AP Biology Points of control The control of gene expression can occur at any step in the pathway from gene to functional protein 1.Packing/unpacking of DNA 2.Transcription 3.mRNA processing 4.mRNA transport 5.Translation 6.Protein processing 7.Protein degradation

55. Regulation of transcription in prokaryotes vs. eukaryotes ProkaryotesEukaryotes Linked genes are organized into clusters known as operons which are under the control of a single promoter. Eukaryotic genes are not organized into operons and each gene requires its own promoter. The genes are primarily regulated by repressors. Regulation by repressors is less common and the primary role of regulation is played by transcriptional activators.

56. Regulation of transcription in prokaryotes vs. eukaryotes ProkaryotesEukaryotes A promoter sequence which controls an operon lies upstream of the operon. Accessory or the regulatory proteins control the recognition of the transcriptional initiation sites by RNA polymerases Those genes which code for a protein have a basic structure consisting of: Exons – Gene sequences which encode for a polypeptide Exons – Gene sequences which encode for a polypeptide Introns – These sequences will get removed from the mRNA before it gets translated. Introns – These sequences will get removed from the mRNA before it gets translated. A transcription initiation site A transcription initiation site Promoter sequences. Promoter sequences. A single operon gets transcribed into a polycistronic mRNA which can be translated into multiple proteins Monocistronic mRNAs which can produce a single polypeptide are produced

57. 57 Transcription factors involved in eukaryotic transcription