Announcements
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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

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.


Announcements

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


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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+


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Outline of Lecture 27

I. Eukaryotic Gene Expression: it’s more complicated being multicellular

II. The Promoter

III. Enhancers

IV. Methylation

V. Alternative splicing


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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 cellNeuron

+ insulin- insulin

- neurotransmitter+neurotransmitter


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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


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Levels of

gene

regulation

***focus today


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The interphase nucleus

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


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Organization/ packaging of DNA

Nucleus= 5-10 m

(0.01mm)

Diploid genome= 6.4x109 bp

0.34nm/bp

DNA=2 meters

(2000 mm)


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Chromatin remodeling


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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


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


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Transcription

factors

TBP-TATA binding protein

TAFs- TATA assoc. factors


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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


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Combinatorial Model of Gene Expression

Liver

Regulatory

TFs increase

transcription

activity

Brain

No reg.TFs in this cell for albumin enhancer


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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.


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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


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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


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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


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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.


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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


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Antirepressor Transcription Factors

Access

Slow txn


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TFs can recruit HATs or HDs


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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


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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?


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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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