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Announcements

Announcements. Tuesday afternoon lab section: lab start time next week is 3pm. 2-3 pm might be a good time to do problem set 6! 2. No advance reading for next week’s lab; focus on your lab report. 3. Problem set 5 due at start of class today. 4. Reading - Ch. 16: skip btm. 442- top 444.

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Announcements

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  1. Announcements Tuesday afternoon lab section: lab start time next week is 3pm. 2-3 pm might be a good time to do problem set 6! 2. No advance reading for next week’s lab; focus on your lab report. 3. Problem set 5 due at start of class today. 4. Reading - Ch. 16: skip btm. 442- top 444.

  2. Review of Last Lecture 1.The lac operon - in detail; know roles of all components • lactose • repressor protein from I gene • Operator sequence • Promoter sequence • Regulation of expression of 3 structural genes: • lacY, lacZ, lacA • CAP + cAMP • Glucose • **Clarification: maximal transcription= repressor bound by lactose and CAP bound to CAP-binding site 2. The trp operon - very briefly

  3. Learning Check • Will B-galactosidase be made: a-always, b-never, c-sometimes (in presence of lactose) 1. I+O+Z+ 2. I+OCZ+ 3. ISO+Z+

  4. Outline of Lecture 27 I. Eukaryotic Gene Expression: it’s more complicated being multicellular II. The Promoter III. Enhancers IV. Methylation V. Alternative splicing

  5. I. Problems of Multicellularity • All of our genes are present in every cell, but only certain proteins are needed. • Expression of a gene at the wrong time, in the wrong type of cell, or in abnormal amounts can lead to deleterious phenotypes or death - even when the gene itself is normal. Pancreatic cell Neuron + insulin - insulin - neurotransmitter +neurotransmitter

  6. Levels of Regulation of Eukaryotic Gene Expression • “What is true of E. coli is only partly true of elephants.” • Prokaryotic control primarily at the transcription level. • Eukaryotic control at several levels

  7. Levels of gene regulation ***focus today

  8. The interphase nucleus Chromosome structure is continuously rearranged, so that transcriptionally active genes are cycled to edge of territories.

  9. Organization/ packaging of DNA Nucleus= 5-10 m (0.01mm) Diploid genome= 6.4x109 bp 0.34nm/bp DNA=2 meters (2000 mm)

  10. Chromatin remodeling

  11. II. Promoters: Eukaryotic vs. Prokaryotic RNA pol II RNA pol Promoters: sequences adjacent to genes, where RNA pol binds to initiate transcription Euk. - Chromatin and TFs Prok. - Naked DNA and no TFs needed

  12. “Promoter-Bashing” Mutations Determine the Critical Regions of DNA for Gene Expression

  13. Transcription factors TBP-TATA binding protein TAFs- TATA assoc. factors

  14. III. Eukaryotic Enhancers and Promoters Promoters- needed for basal level transcription Enhancers- needed for full level transcription; location and orientation variable Two types of transcription factors bind enhancers and affect levels of txn: true activators and anti-repressors

  15. Combinatorial Model of Gene Expression Liver Regulatory TFs increase transcription activity Brain No reg.TFs in this cell for albumin enhancer

  16. Binding of True Activator TFs to Enhancers Greatly Stimulates Transcription Looping of DNA allows Activator TF bound to Enhancer to interact with Promoter, facilitating binding of Basal TF complex.

  17. Types of Regulatory Transcription Factors • True activators are modular proteins: one domain binds DNA in enhancer; one domain interacts with protein at promoter • Classified by DNA-binding motifs in the protein: • Helix-Turn-Helix • Zinc Finger • Leucine Zipper • Helix-Loop-Helix

  18. Helix-Turn-Helix (HTH) TFs • Two -helical stretches of AA’s linked by non-helical sequence of AA’s • e.g. lac repressor protein (binds operator DNA) • also eukaryotic homeodomain TFs, important in embryonic development

  19. Zinc Finger TFs • Zn2+ is coordinated between His and Cys residues in protein, forming “finger” of protein • 2-13 fingers may be present • Found in pseudooncogenes and in DrosophilaKruppel TF in embryonic development

  20. Leucine Zipper TFs • Dimeric, held together by interactions between leucine residues • includes AP-1 TF, a heterodimer of Jun and Fos proteins, important in control of cell division; mutation can cause cancer.

  21. Helix-Loop-Helix (HLH) TFs • Dimeric: two subunits of two helices linked by loops • includes mammalian TFs for muscle differentiation - MyoD, myogenin and Myf-5

  22. Antirepressor Transcription Factors Access Slow txn

  23. TFs can recruit HATs or HDs

  24. IV. Control of gene expression by DNA Methylation • Addition of CH3 to selected C’s in DNA can inactivate genes, e.g. high levels are seen in inactivated X chromosome of female mammals. • Mammals have about 5% methylation. • Not essential in eukarotyes, since Drosophila has 0% methylation. • First observed in lac operon: methylation of operator DNA sequence affects binding by repressor

  25. V. Post transcriptional gene regulation If humans have approximately the same number of genes as a fruit fly, and we require more complex cellular functions (presumably with a larger number of proteins) - how do we accomplish this?

  26. Alternative splicing 1. Chromosomal ratio activates txn of Sxl in females only 2. SXL controls splicing of tra-2 mRNA 3. Females: exon 2 (which has a stop codon) is removed via SXL Males: exon 2 is not removed. Males: no active TRA Females: TRA is made. 5. TRA directs splicing of dsx mRNA in specific manner; in males default splicing occurs.

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