Alternate sigma factor usage: controls selective transcription

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Ways to Regulate Transcription
Alternate sigma factor usage controls selective
transcription
of entire sets of genes
vegetative (principal s)
1
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(16-19 bp)
(5-9 bp)
TTGACA
TATAAT
A
heat shock
1
s32
(13-15 bp)
(5-9 bp)
CNCTTGA
CCCATNT
A
1
nitrogen starvation
s60
(6 bp)
(5-9 bp)
TTGCA
CTGGNA
A
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The pathway of gene expression
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Pre-mRNA
microRNAs (miRNA) RNA molecules of about 2123
nucleotides in length, which regulate gene
expression. are processed from primary
transcripts known as pri-miRNA.
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5UTR
3UTR
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PolyA RNA in the nucleus and cytoplasm in two
human cell lines
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Eukaryotic Promoters

• More complex and diverse than prokaryotic
  promoters
• RNA polymerase I promoters
• Multiple copies of rRNA genes exist
• RNA polymerase I recognizes only one
  species-specific promoter
• Requires a core promoter element and an upstream
  promoter element
• RNA polymerase III promoters have variable
  locations relative to the transcribed gene
• More attention has been paid to RNA polymerase II
  promoters because they are involved in the
  transcription of mRNA

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RNA polymerase
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E
S
RE
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• Initiation
• Elongation
• Termination

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TATA-Binding Protein (TBP)

• Formation of the preinitiation complex (PIC)
  begins when TBP protein of TFIID binds the TATA
  box of a promotor
• DNA binds the concave surface in a sharply bent
  conformation
• Double helix is partially unwound and minor
  groove widens

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TFII transcription factor of RNA PolII
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????? ?-INITIATION
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Initiation complex
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Expanding the functions of RNA polymerase
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Stages in Initiation of Transcription

• Bacterial transcription
• Closed complex holoenzymepromoter
• Open complex (DNA melting, not need ATP)
• Abortive transcripton
• Productive initiation
• Transcribe past 9
• Sigma dissociates
• Elongation

• Eukaryotic transcription
• Preinitiation complex (PIC) assembly
• PIC activation (DNA melting, needs ATP)
• Abortive transcription
• Productive initiation
• CTD phosphorylated
• Promoter clearance
• Elongation

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Structure of RNA Polymerase II

• RNA polymerase II from yeast has been extensively
  characterized
• Contains two large subunits that are homologs of
  prokaryotic RNA polymerase subunits b and b
• Contains 10 smaller subunits, including homologs
  of a and r
• Structural features
• Thumb
• DNA-binding channel
• Channel for single-stranded RNA

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3-dimensional view of yeast RNA Pol II
Both yeast RNA Pol II and E. coli RNA polymerase
core Have a similar shape and have the channel
for DNA template.
RNA polymerase II from yeast has been extensively
characterized Contains two large subunits that
are homologs of prokaryotic RNA polymerase
subunits b and b Contains 10 smaller subunits,
including homologs of a and r Structural features
- Thumb - DNA-binding channel - Channel for
single-stranded RNA
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Parallels between initiation pathway in
prokaryotes and eukaryotes
From Eick et al. (1994) Trends in Genetics 10
292-296
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InitiationAssembly of the PIC

1. TBP component of TFIID binds the TATA box
2. TFIIA and TFIIB bind
3. TFIIF binds RNA polymerase and escorts it to the
   complex
4. TFIIE and TFIIF complete the PIC

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initiation
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INITIATION

• Prokaryotes RNA polymerase
• Binds to promoter
• Pulls 2 strands apart
• Eukaryotes Transcription initiation complex
• Transcription factors
• Proteins
• Bind to the promoter
• RNA polymerase

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Elongation
NTPs
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Elongation
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RNA polymerase II
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TERMINATION

• RNA polymerase meets the terminator
• Terminator sequence AAUAAA
• RNA polymerase releases from DNA
• Prokaryotes-releases at termination signal
• Eukaryotes-releases 10-35 base pairs after
  termination signal

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polyadenylation
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Initiation, Elongation, and Termination

• Helicase activity of TFIIF promotes DNA unwinding
• Kinase activity of TFIIF phosphorylates RNA
  polymerase, causing a conformational change in
  the polymerase that initiates transcription
• TFIIA, TFIIB, and TFIID (including TBP) remain
  bound to the promoter to aid in PIC reassembly
• TFIIH and TFIIE are released
• Activity of polymerase is enhanced by proteins
  called elongation factors
• Termination mechanism is not well understood
• Polymerase is dephosphorylated and released

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Enhancers and Silencers

• The activities of many promoters in eukaryotes
  are increased by enhancers and decreased by
  silencers
• Can be upstream, downstream, or in the midst of a
  transcribed gene
• Activators or repressors can bind to enhancers
  and silencers to influence RNA polymerase binding
  to promoters
• Enhancers and silencers act only in cells that
  contain the appropriate activator or repressor
  proteins

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Transcription at Other Promoters

• RNA polymerase II promoters that lack TATA boxes
  require several of the same transcription factors
  that initiate transcription from TATA boxes,
  including TBP
• Transcription by RNA polymerase I and RNA
  polymerase III requires different sets of
  transcription factors, but TBP is required in all
  cases
• Studying transcription initiation is difficult
  because transcription factors are present at very
  low concentrations

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TBP is used by all 3 RNA polymerases

• TBP is a subunit of an important GTF for each of
  the 3 RNA polymerases
• TBP or TFIID for Pol II
• SL1 for Pol I
• TFIIIB for Pol III
• It does NOT always bind to TATA boxes promoters
  for RNA Pol I and Pol III (and even some for Pol
  II) do not have TATA boxes, but TBP is still
  used.
• The GTFs that contain TBP may serve as
  positioning factors for their respective
  polymerases.

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RNA Pol I
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Signal transduction of Pol I
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Transcription complex
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transcription
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polyA-PolII termination
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Transcription regulation
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Upstream control element
Upstream binding factor
Transcription associated factor
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Gene expression
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Basics of cell biologydevelopment
Fertilized egg
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Basics of cell biologydevelopment
Cells differentiation is due to different
gene Switches going on and off
These cells are different because they express a
subset of their 24,000 genes
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To specify a new cell place it in the right
environment or
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EGF
Hedgehog
Wnt
Retinoic acid
FGF
HGF
To specify a new cell place it in the right
environment or Substitute drugs/proteins for
environment Mophogens
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Chromosome localization in interphase
In interphase, chromosomes appear to be localized
to a sub-region of the nucleus.
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Domainopening is associated with movement to
non-hetero-chromatic regions
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Proposed sequence for activation

• 1. Open a chromatin domain
• Relocate away from pericentromeric
  heterochromatin
• Establish a locus-wide open chromatin
  configuration
• General histone hyperacetylation
• DNase I sensitivity
• 2. Activate transcription
• Local hyperacetylation of histone H3
• Promoter activation to initiate and elongate
  transcription

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From silenced to open chromatin
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Movement from hetero- to euchromatin
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Nucleosoe remodelers and HATs further open
chromatin
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Assembly of preinitiatin complex on open chromatin
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Transcription factor binding to DNA is inhibited
within nucleosomes

• Affinity of transcription factor for its binding
  site on DNA is decreased when the DNA is
  reconstituted into nucleosomes
• Extent of inhibition is dependent on
• Location of the binding site within the
  nucleosome.
• binding sites at the edge are more accessible
  than the center
• The type of DNA binding domain.
• Zn fingers bind more easily than bHLH domains.

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Stimulate binding of transcription factors to
nucleosomes

• Cooperative binding of multiple factors.
• The presence of histone chaperone proteins which
  can compete H2A/H2B dimers from the octamer.
• Acetylation of the N-terminal tails of the core
  histones
• Nucleosome disruption by ATP-dependent remodeling
  complexes.

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Binding of transcription factors can destabilize
nucleosomes

• Destabilize histone/DNA interactions.
• Bound transcription factors can thus participate
  in nucleosome displacement and/or rearrangement.
• Provides sequence specificity to the formation of
  DNAse hypersensitive sites.
• DNAse hypersensitive sites may be
• nucleosome free regions or
• factor bound, remodeled nucleosomes which have an
  increased accessibility to nucleases.

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Chromatin remodeling ATPases catalyze stable
alteration of the nucleosome
II form a stably remodeled dimer, altered DNAse
digestion pattern III transfer a histone octamer
to a different DNA fragment
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Covalent modification of histone tails
N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H3
4
9 10
14
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27 28
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N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H4
5
8
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16
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acetylation
phosphorylation
methylation
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Yeast SAGA interacting with chromatin