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Lecture 8 Transcription Initiation Prokaryotic Eukaryotic Reading: Chapter 4 (115-116) Chapter 11...
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Transcript of Lecture 8 Transcription Initiation Prokaryotic Eukaryotic Reading: Chapter 4 (115-116) Chapter 11...
![Page 1: Lecture 8 Transcription Initiation Prokaryotic Eukaryotic Reading: Chapter 4 (115-116) Chapter 11 Molecular Biology syllabus web siteweb site.](https://reader037.fdocuments.us/reader037/viewer/2022103015/55168e79550346a25b8b4df5/html5/thumbnails/1.jpg)
Lecture 8
Transcription InitiationProkaryotic
Eukaryotic
Reading:
Chapter 4 (115-116)
Chapter 11
Molecular Biology syllabus web site
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Bacterial transcription initiation
• RNA polymerase initiates transcription of most genes at a unique DNA position lying upstream of the coding sequence
• The base pair where transcription initiates is termed the transcription-initiation site or start site
• By convention, the transcription-initiation site in the DNA sequence is designated +1, and base pairs extending in the direction of transcription (downstream) are assigned positive numbers which those extending in the opposite direction (upstream) are assigned negative numbers
• Various proteins (RNA polymerase, activators, repressors) interact with DNA at or near the promoter to regulate transcription initiation
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Differences in E. coli promoter sequences affect the frequency of transcription initiation
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Lac Operon
• Beta Galactosidase (lacZ)
• Permease (lacY)
• Transacetylase (lacA)
lacZ lacY lacA
Lac promoter
mRNA
protein
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LacZ (-galactosidase)
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Eukaryotes
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Promoter deletions to identify transcriptional control sequences
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Promoter deletions to identify transcriptional control sequences
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Run-off transcription: to map transcription initiation sites
METHOD
1. Isolate nuclei from specific tissues
2. Incubate: nuclear extract, restriction fragments containing start site, 32P-UTP
3. Synthesized RNA is separated by electrophoresis
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Common Eukaryotic Promoter Consensus: TATA box
Alternate promoters:initiators- degenerate consensusGC-rich region (giving rise to multiple start sites)
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Run-on transcription assay rate of transcription initiation
METHOD
1. Isolate nuclei from specific tissues
2. Label growing RNA chains with 32P-UTP
3. Hybridize to filters containing test cDNAs
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S1 nuclease
mRNA
32P-labelled DNA probe
mRNA
32P-labelled DNA probe
denature
gel electrophoresis
S1 protection to assay transcription initiation rates
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Enhancer elements
cis-acting sequences that serve to increase rate of transcription initiation regardless of their location near the gene (first discovered in SV40 viral genome).
S1 Protection Assay: to test rate of transcription initiation
METHOD
1. Transfect construct with/without enhancer into test cells
2. Isolate RNA and hybridize with 32P-labeled DNA probe
3. Treat with S1 nuclease, denature, subject to gel electrophoresis
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Typical eukaryotic gene
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Column purified fractions of DNA-binding proteins incubated with test promoter fragment
Gel-shift assays identify general region of protein-DNA interaction
METHOD
1. Isolate nuclei and prepare protein extract or purify DNA binding proteins
2. Incubate with restriction fragment or short DNA
3. Separate by agarose gel electrophoresis
Controls: non-specific DNA; no extract
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METHOD
1. Isolate restriction fragment
2. Add DNA-binding protein
3. Partially digest with DNaseI
4. Separate by polyacrylamide gel electrophoresis
Controls: no protein
The footprint of RNA polymerase and lac repressor on the lac control region
DNase I footprinting assays identify specific regions of protein-DNA interactions
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The lac control region contains three critical cis-acting sites
Figure 10-9
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How to demonstrate the specificity of a putative transcription factor?
• In vitro
• In vivo
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• In vitroMETHOD
1. Set up in vitro transcription reaction with DNA template, 32P-UTP, plus or minus transcription factor
2. Separate transcripts by gel electrophoresis
Controls: non-specific DNA; no protein
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• In vivo
METHOD
1. Prepare two constructs:
a) encoding transcription factor X
b) reporter gene downstream of transcription factor binding site plus minimal promoter
2. Transfect cells and assay for reporter gene activity
Controls: transfect each plasmid alone
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Transcription factors have separate domains
•DNA binding
•Transcription activation
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Yeast Two-hybrid System- to fish out interacting transcription factors
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