Prokaryotes have only one RNA polymerase

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Prokaryotes have only one RNA polymerase
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Purified RNA polymerases use different templates
to synthesize RNAs
Salt conc.
Metal ions
Location
(45S)
low Mg2 Nucleus middle Mn2
Nucleolus Broad Mn2 Nucleolus range
(heterogeneous nuclear)
(small nuclear)
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Robert Roeder and Bill Rutter showed that cell
extracts contain at least three different RNA
polymerase activities by subjecting cell extracts
from sea urchin embryos to ion-exchange
chromatography, which separates proteins by their
charges.
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Eukaryotes - Three RNA Polymerases
?-amanitin
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Identification of RNA polymerase subunits
Co-immunoprecipitation Assay/ Epitope Tagging
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Promoters
Class I (Pol I promoter for rRNA precursor gene)
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Class II
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• TATA box locates the start of transcription 30
  bp downstream
• TATA box sometime is important for the efficiency
  of transcription
• TATA-binding protein (TBP) binds to TATA-box and
  initiates the assembly of transcription factors
  and RNA polymerase

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Initiator --PyPyANT/APyPy -- is sufficient by
itself to direct transcription Downstream
promoter elements (DPE) -- quite common in
fruitfly genes. Found 30 bp downstream of the
transcription initiation site Have the consensus
sequence -- G(A/T)CG -- behave as a TATA-box BRE
-- (G/C)(G/C)(G/A)CGCC -- TFIIB-binding site
Upstream element
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Class III (Pol III promoter)
Unlike class I and II promoters, classical class
III promoters are located within the genes.
U6 snRNA, 7SL, and 7SK RNA gene still need
elements 5 of the genes. Their promoters
resemble class II promoters (nonclassical class
III promoter).
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Class II preinitiation complex
Six class II factors TFIIA, TFIIB, TFIID, TFIIE,
TFIIF and TFIIH. Preinitiation complex is
assembled in a orderly manner
D
DA
DAB
DAB-PolF
DAB-PolF-EH
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Initiation, Promoter Clearance, and Elongation
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Two important assays to examine protein-DNA
interaction DNase I footprint analysis Gel
mobility shift assay or Gel retardation assay
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DNase I footprint assay
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Mobility shift assay Gel retardation assay
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TFIID
TFIID binds to TATA box TFIID is composed of
TATA-box-binding protein (TBP) and 8-10
TBP-asscociated factors (TAFs) TBP is a highly
conserved protein (from yeast to humans). The
C-terminal 180 amino acid of TBP is the
TATA-binding domain.
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TBP is Present in ALL Transcription Complexes
regardless of their TATA content, or the nature
of polymerase