announcements 1. tuesday afternoon lab section: lab start time next week is 3pm. 2-3 pm might be a...
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Announcements
1. 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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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 cell Neuron + 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 generegulation
***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 TFsProk. - 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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Transcriptionfactors
TBP-TATA binding proteinTAFs- TATA assoc. factors
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III. Eukaryotic Enhancers and Promoters
Promoters- needed for basal level transcriptionEnhancers- 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
LiverRegulatoryTFs increasetranscriptionactivity
BrainNo 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 Drosophila Kruppel 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 SXLMales: exon 2 is not removed.
4. Males: no active TRAFemales: TRA is made.
5. TRA directs splicing of dsx mRNA in specific manner; in males default splicing occurs.