Ch 10. Multiple forms of Eukaryotic RNA Polymerase

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Ch 10. Multiple forms of Eukaryotic RNA
Polymerase
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Regulation of Gene expression

• Regulatory DNA sequence
• Gene regulatory protein

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Transcription
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Separation of eukaryotic RNA polymerase
Multiple forms of Eukaryotic RNA Polymerase
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Cellular localization of the three RNA polymerases
Nucleolus pol I - rRNA Nucleoplasm pol II
mRNA pol III tRNA, 5S
rRNA
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Roles of Eukaryotic RNA polymerases
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Sensitivity of RNA polymerases to a-amanitin
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RNA polymerase subunits (S. cerevisiae)
b b

• Complex
• 2 large subunit others
• 5 common subunits

a
a
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Human RNA polymerase II
By R. Young, 2000
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Subunits of yeast RNA polymerase
Immunoprecipitation
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Yeast RNA Polymerase II subunits

• Core subunit Rpb1, 2, 3 ???? RNA pol
  subunit (b, b, a) ? ??
• Common subunit Rpb 5, 6, 8, 10, 12 ?? ?????
  ??
• Nonessential subunit Rpb 4, 9
• Non-classified Rpb 7, 11

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Different forms of Rpb1 subunit
RNA pol IIA bind to promoter RNA pol IIO (CTD
phosphorylation) elongation
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Structure of RNA polymerase
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Crystal structure of RNA polymerase
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Structure of RNA polymerase containing DNA
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Promoters

• Three polymerase recognize different promoters
• Regulatory DNA sequence
• Gene regulatory protein

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Class II promoter
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Effect of deletions in the SV40 early promoter
TATA box
TATAAA(A/T) -25 upstream from initiation
site TATA-less housekeeping gene, - GC box, DPE
Developmentally regulated
genes (Homeotic gene, Immune system)
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TATA box locate the start site of transcription
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Linker scanning mutagenesis
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Effects of linker scanning mutations in the
promoter
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• Initiator (Inr) PyPyANT/APyPy
• Downstream promoter element (DPE) in Drosophila
• 30 bp downstream of the Tc initiation site
• G(A/T)CG
• TATA-less promoter
• bind to TFIID
• TFIIB recognition elements (BREs)
• TFIIB for preinitiation complex
• (G/C)(G/C)(G/A)CGCC

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Effects of mutations in GC Boxes on promoter
activity
Upstream elements
GC box Sp1 binding site, orientation-independenc
e, position-dependent CCAAT (cat) Box CTF
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Class I promoter (rRNA promoter )
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5S rRNA promoter (Class III promoter)
Internal promoter classical class III promoter
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Promoters of polymerase III genes
U6
( Non-classical 7SK RNA promoter, a-amanitin?
?? ???)
Hybrid (type II type III ) 7SL RNA
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7SK RNA promoter non-classical class III
promoter
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SV40 promoter and enhancer
Enhancer Silencer

• Cis-acting element
• Position-independent
• Orientation-independent

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Effect of deletion in Ig enhancer
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Enhancer is Position-and Orientation-independent