Molecular Biology

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Molecular Biology

• Part II The Central Dogma
• Transcription

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DNA does nothing without proteins

• How do we know when proteins interact with DNA?
• to interact, molecules have to stick together
• Some more MM three tests for proteins sticking
  to DNA
• 1. filter binding assay
• 2. mobility shift assay
• 3. DNA footprint assay

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protein
radiolabel DNA

• Nitrocellulose
• filter binds
• protein
• ssDNA
• not dsDNA

Filter retains radioactivity if protein binds to
DNA
DNA-protein binding assay 1 Filter-binding assay
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Indirect binding second protein will shift DNA
mobility only when first protein is present
DNA-protein binding assay 2
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Overview

• 1. Initiation
• 2. Elongation
• 3. Termination

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Lewin 1990 Genes IV
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RNA polymerase

• Holoenzyme core s (sigma)

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1. labeled T7 DNA enzyme
Holoenzyme sticks tight to DNA, so radioactivity
is retained on filter
2. then add excess of unlabeled T7 DNA (picks up
enzyme when it leaves labeled DNA)
Core enzyme has loose binding, so radiolabeled
DNA is released and not retained by filter
3. time course shows strength of DNA- protein
binding (filter-binding assay)
Fig. 6.5 Sigma stimulates tight binding between
RNA polymerase and promoter.
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Promoters

• consensus sequences
• TATA box
• startpoint
• -35 box
• Directed mutation analysis
• Footprinting

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(No Transcript)
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Felsenfeld 1985 Scientific American
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Assay 3 DNAse footprinting locates protein
binding
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Lewin 2000 Genes VII
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Stereo views of RNA pol core from T. aquaticus
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Catalytic center of RNA pol from T. aquaticus
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Termination

• ? (rho)-independent
• ?-dependent

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Watson et al. 1987 Molecular Biology of the
Gene 4th ed.
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?-dependent terminator has hairpin but no
poly-U sequence
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In vitro transcription
Without ?
With ?
rho has no effect on the size of previously made
transcripts, but reduces the size of
transcripts made in its presence
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Without ?
Note size shift from DNA binding to RNA when rho
is absent
RNA
DNA
With ?
RNA made in the presence of rho sediments
independently of DNA without rho, DNA
RNA sediment as larger stuff, connected by
transcription apparatus. Rho separates RNA from
DNA.
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