Class I factors: There Are Only Two

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Class I factors There Are Only Two
Core binding factor SL1 in human
Isolated in 1985 as one of two fractions needed
for transcription in vitro from a human rRNA
template
Composed of three TAFs (TAFI110, TAFI63, and
TAFI48) and TBP
Upstream binding factor UBF in human
Probably only one polypeptide 97 kDa
Binds to upstream promoter element (UPE) to
enhance initiation
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Class III Factors Three Identified
TFIIIA
First eukaryotic transcription factor identified
First DNA-binding protein found to contain zinc
fingers (has 9)
Binds specifically to internal promoters of 5S
rRNA genes
TFIIIB and TFIIIC
Both required for transcription and depend on
each other
TFIIIB contains TBP
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Common Themes in Eukaryotic Transcription
Initiation
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Key Points
1. Initiation of eukaryotic transcription
involves the formation of large complexes at the
promoter. These complexes generate tremendous
versatility.
2. Of the three transcription classes, class II
is the most complicated but also the best
understood. At least six factors are required,
most of which are complexes of several
polypeptides.
3. A common theme is that TBP plays an important
role. It can be involved in initiation either by
binding to the TATA sequence of by associating
with other factors.
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Transcription Activation in Eukaryotes
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Transcription Activation in Eukaryotes
A. Transcriptional activators categories and
structures
B. Functional properties of activators
C. Regulation of activators
D. Another level of regulation chromatin
remodeling
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A Transcription Factor Factory
Tjian lab, 1986
Kadonaga, Cell (2004) 116, 247-257
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Activators Have Two DomainsDNA Binding and
Activation Domains
DNA Binding
Activation
Zn-containing modules
Acidic domains
Homeodomains
Glutamine-rich domains
bZIP and bHLH domains
Proline-rich domains
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DNA Binding Domains
DNA Binding
Zn-containing modules
Homeodomains
bZIP and bHLH domains
First identified in transcription factor TFIIIA
NMR used to solve first structure (1989)
Some transcription factors have multiple Zn
fingers. Zif268 from mouse has three. Residues
from each ?-helix make sequence-specific contacts
in the major groove (1991).
Zn fingers bind with sequence specificity, but
no simple code exists to predict the DNA sequence
that a given zinc finger motif will recognize.
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DNA Binding Domains
DNA Binding
GAL4 DNA binding domain
Zn-containing modules
Controls a set of genes responsible for
galactose metabolism
Homeodomains
Binds to DNA as a dimer formed by coiled-coil
interaction
bZIP and bHLH domains
Each monomer binds two Zn ions
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DNA Binding Domains
DNA Binding
Members of helix-turn-helix family
Discovered in regulatory genes of Drosophila
Zn-containing modules
Helix 3 occupies major groove and an extension
of helix 1 occupies minor groove
Homeodomains
bZIP and bHLH domains
T. Kornberg, C. Pabo and colleagues, 1990
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DNA Binding Domains
DNA Binding
bZIP dimer forms a leucine zipper
coiled-coil. Each helix contacts DNA in the major
groove.
Zn-containing modules
Homeodomains
b for basic region, which contacts DNA
ZIP for the dimerization (zipper) region
consisting of amphipathic helices that form
parallel coiled-coil interactions
bZIP and bHLH domains
Structures of GCN4 Yeast protein that
up-regulates amino acid biosynthesis
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DNA Binding Domains
DNA Binding
Zn-containing modules
Homeodomains
bZIP and bHLH domains
MyoD Involved in muscle development
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DNA Binding and Activation Domains Seem to
Function Independently
Activation can be obtained from artificial
fusions of DNA binding and activation domains
Activation domain of GAL4 fused to DNA binding
domain of prokaryotic repressor LexA, ability to
activate transcription was assayed in yeast
(Brent Ptashne)
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Transcription Activation in Eukaryotes
A. Transcriptional activators categories and
structures
B. Functional properties of activators
C. Regulation of activators
D. Another level of regulation chromatin
remodeling
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Functional Properties of Activators
Recruitment of RNAP II by interactions with
TFIID Herpes virus transcription factor VP16
(1990)
Transcription indicates binding of TFIID to
immobilized VP16
Direct or indirect interaction?
Involvement of mediator and perhaps other
complexes?
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Dimerization of Activators Can Tighten DNA Binding
Some form homodimers, and others form
heterodimers
Example of heterodimer is Jun-Fos, both are
members of the bZIP family
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How Close Does An Activator Have to Be to the
Promoter?
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How Close Does An Activator Have to Be to the
Promoter?
Catenane created by including phage lambda
recombination sites on plasmid (1989)
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This Mechanism Allows For Multiple Interactions
With Activators
Human metallothionine gene has sites for
several activators
Leads to concept of enhancer as a region that
can bind any of a number of activator proteins
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What About Activators With Sites Too Close to the
Promoter to Allow Looping Out of the DNA?
DNA bending established for some proteins with
HMG domain.
HMG-containing proteins do not themselves
activate transcription, but the bending allows
formation of an optimal enhanceosome with other
activators.
Method was to evaluate gel migration and vary
position of protein binding site within short
DNAs.
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What Prevents Enhancer Regions From Activating
the Wrong Gene? Insulators
Sequence elements that bind proteins to
insulate genes from effects of more distant
activators and repressors
Mechanism of activity is not yet clear
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Transcription Activation in Eukaryotes
A. Transcriptional activators categories and
structures
B. Functional properties of activators
C. Regulation of activators
D. Another level of regulation chromatin
remodeling
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What Turns Transcription Factors On and Off?
Activation by transport to the nucleus (signal
transduction pathways)
Covalent modification, frequently the end of
signal transduction pathways -
Phosphorylation - Ubiquitination -
Sumoylation - Methylation - Acetylation
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Signal Transduction Pathway Ending in
Transcription Factor Phosphorylation
Promotes cell division
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Attachment of the Polypeptides Ubiquitin or SUMO
Inactivates Transcription Factors
Ubiquitination targets proteins for destruction
by the proteosome
However, in some cases attachment of a single
ubiquitin can increase activity. An example of
this is the tumor suppressor protein p53
Attachment of SUMO (small ubiquitin-related
modifier) targets factors to specific nuclear
compartments where they are sequestered away from
their target genes
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Transcription Activators Can Also Be Acetylated
or Methylated
All of these modifications occur on lysine
residues (K)
One modification will inhibit others on a given
lysine, but can favor additional modification on
nearby lysines
Freiman and Tjian, Cell (2003) 112 11-17
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Transcription Activation in Eukaryotes
A. Transcriptional activators categories and
structures
B. Functional properties of activators
C. Regulation of activators
D. Another level of regulation chromatin
remodeling
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Chromatin Provides Another Level of
Transcriptional Regulation
Chromatin remodeling complexes - SWI/SNF (first
complex identified) - ISWI - NuRD - INO80 All
complexes include an ATPase
Kornberg, R, PNAS 104, 12955 (2007)
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Mechanism of Chromatin Remodeling
Mechanistic study used single nucleosome and
ATPase component of SWI/SNF (2001)
Remodeling followed by availability of
different restriction sites in DNA
Results suggest multiple remodeled complexes
and an element of randomness to remodeling process
Fast
Fast
Slow
Fast
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Remodeling in the Human IFN-? Gene The Histone
Code
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Remodeling in the Human IFN-? Gene The Histone
Code
Histone modifications appear in a sequence
Certain acetylations increased binding of
SWI/SNF component (not shown)
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Remodeling in the Human IFN-? Gene The Histone
Code
Histone modifications appear in a sequence
Certain acetylations increased binding of
SWI/SNF component (not shown)
Leads to model in which initial modifications
lead to remodeling, which allows formation of
preinitiation complex
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Key Points
1. Activator proteins are required for efficient
transcription in eukaryotes. They consist of two
separable domains a DNA-binding domain and an
activator domain.
2. Activation an occur even from a large
distance. The intervening DNA is most likely
looped out, allowing the activator to contact a
component of the general transcription machinery.
3. Regulation of activators occurs by covalent
modification, such as phosphorylation,
ubiquitination, sumoylation, methylation, and
acetylation. These are frequently part of signal
transduction pathways.
4. An additional level of regulation occurs at
the level of chromatin. Chromatin remodeling
complexes act in conjunction with histone
modifications and via incompletely understood
mechanisms to expose DNA to the transcription
machinery.
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