Class I Promoters for nRNAP I

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Class I Promoters (for nRNAP I)

• Sequences less conserved than Class II
• Usually 2 parts
• UCE upstream control element , -150 to -100 in
  human rRNA
• Core from - 45 to 20
• Spacing between elements also important

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Results of Linker Scanning mutagenesis of the
human rRNA promoter.
The DNA was transcribed in vitro, and the
efficiency is expressed relative to the wild-type
promoter.
Fig. 10.24
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Figure 10.25
Effect of Spacing of Two rRNA promoter Elements
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Class III promoters (for nRNAP III)

• 2 types
• Internal promoters
• - 5S rRNA (Box A, Intermediate element, Box C)
• - tRNA (Box A, B)
• Class II like promoters
• - contain TATA box
• - 7SL gene, promoter is upstream of coding region

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Fig. 10.28
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Fig. 10.26
Effect of deletions at the 5-end of the Xenopus
5S rRNA gene on its transcription in vitro.
Result It required deleting more than 50 bp into
this small gene to knockout its transcription.
Conclusion the promoter for the 5S rRNA gene is
internal!
Numbers at bottom of lanes are the bp deleted.
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How can you determine the 3-end of the promoter?
Used cordycepin (3-ATP) to stop transcription as
3 deletion would remove terminator sequence
Fig. 10.27a
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Fig. 10.27b Effect of 3 Deletion on 5S rRNA
Gene Transcription
Note the difference between f and g
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Further characterization showed 3 boxes in 5S
rRNA promoter
Fig. 10.28
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How was the promoters for Class III genes with
Polymerase II-like promoters discovered?
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Figure 10.29
Fig. 10.29 Effect of 5-Deletion on 7SK RNA
Promoter
Cloned into two vectors Stops if -26 to -15
deleted
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Enhancers and Silencers

• Enhancers stimulate transcription, silencers
  inhibit.
• Both are orientation independent.
• Flip 180 degrees, no effect
• Both are position independent.
• Can work at a distance from promoter
• Enhancers have been found all over
• Bind regulated transcription factors.

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An enhancer in an intron of a gamma-globulin gene.
En deleted
FL DNA
No DNA
Fig. 10.32
(a) Genes were constructed with the enhancer
inverted (B), with it moved upstream of the gene
(C) and inverted (D). The DNAs were transfected
into mouse cells and synthesis of the protein was
assessed by pulse-labeling with a radioactive
amino acid, immunoprecipitation, and separation
by SDS-PAGE and autoradiography.
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What is the effect of histones on transcription
in vitro?

• Assemble core histones on a plasmid (1/200 bp),
  nucleosomes inhibit transcription by blocking
  promoter binding sites.
• Addition of H1 further represses transcription
  (by binding to the linker DNA), but this can be
  overcome by activators such as Sp1.
• There are regulatory proteins, such as the
  glucocorticoid-receptor complex, that can
  remove histones from certain promoters.

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2 Models for Transcriptional Activation
Nucleosome covers promoter, still repressed after
H1 removed. Remove nucleosome with special
factors.
H1 (yellow) covers promoter, remove it and bind
activators (factors).
Fig. 13.24
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Figure 13.17
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In Vivo Studies

• Promoters of active genes are often deficient in
  nucleosomes

SV40 virus minichromosomes with a
nucleosome-free zone at its twin promoters.
Can also be shown for cellular genes by DNase I
digestion of chromatin promoter regions are
hypersensitive to DNase I.
Fig. 13.25
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Two Complexes Play Major Roles
HDAC - Histone Deacetylases HAT - Histone Acetyl
Transferase
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Figure 13.4c
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Histone acetylation

• amino groups of lysine side chains
• unacetylated histones tend to repress
  transcription
• acetylated histones tend to activate
  transcription
• Histone acetyl transferase (HAT)Histone
  deacetylase

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Acetylation continued

• Acetylation of histone tails neutralizes some of
  the positive charge, causing them to relax their
  grip on the DNA.
• Reduces nucleosome cross-linking. That is the
  interaction between histones in neighboring
  nucleosome. (eg. H4 in one nucleosome and H2A-H2B
  dimer in the next one.

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Acetylation continued

• Also some TFs recognize acetylated histones. eg.
  TAFII250 has a double bromodomain and recognizes
  low level acetylated histones. Once bound it is a
  HAT and increases acetylation.

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Figure 13.29
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Proposed Mechanism of Histone Deacetylation and
Hyperacetylation in Yeast Transcription
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Models for euchromatic or heterochromatic
histone tail modifications
T. Jenuwein et al., Science 293, 1074 -1080
(2001)
Published by AAAS
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Models for euchromatic or heterochromatic
histone tail modifications
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Translating the "histone code."
T. Jenuwein et al., Science 293, 1074 -1080
(2001)
Published by AAAS
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Figure 13.35
HAT
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What about elongation by RNA polymerase?

• How does RNA polymerase transcribe through
  regions with histones/nucleosomes?
• 2 possibilities
• 1. It could partially open nucleosomes and slide
  around the DNA, which is on the outside.
• Or
• 2. It could completely displace nucleosomes.

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Experimental Strategy

• Construct DNA with only 1 nucleosome on it,
  downstream of a promoter.
• Transcribe DNA in vitro.
• Determine if the nucleosome moves to a new
  position.
• DNA specifically bound in a nucleosome can be
  recovered after Micrococcal Nuclease digestion
  which degrades all DNA not protected by the
  nucleosome.

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DNA protected by the nucleosome core is recovered
after micrococcal nuclease digestion,
radiolabeled, and then hybridized to restriction
enzyme digests of pB18.
Conclusion some of the nucleosome (blue oval) is
repositioned on pB18 after transcription in vitro
with the viral RNA polymerase.
(a) Restriction map of pB18, and (b) expected DNA
fragments with different enzyme combinations.