Control of Gene Expression 1: Transcriptional Controls (lecture 9) Flashcards
if skin cells are isolated from adult frogs and the nucleus implanted into a de-nucleated cell then a _____ embryo is formed
normal
Different cells contain the same genes about _____ in humans but they express different sets of ___
25,000 in humans, proteins
Each cell has the same genome, but differentiation in cells depends on
changes in gene expression
Common proteins are called _______ what is an example
housekeeping proteins, glucose metabolism
what is an example of a specifically limited protein
hemoglobin
The typical human cell expresses _____ of its 25,000 genes but level of gene expression varies. What is an example
30-50%, ex. fingerprint expression profiles, microarrays or RNA sequence
What are some of the ways to control gene expression
- transcriptional control
- RNA processing control
- RNA transport and localization control
- Translation control
- mRNA degradation control
- translation control
- protein activity control
Gene regulation requires
- short stretch of DNA of defined sequence- recognition sites for DNA binding proteins
- Gene regulatory proteins- transcription factors that will bind and activate gene
Recognition sequences for regulatory proteins ex.
GATA1: TGATAG
Heterodimers are often formed from tow different transcription regulators. Transcription regulators may form heterodimers with more than one patterned proteins, the same transcription regulator can be “reused” to breast several
distinct DNA-binding specificities
What does the “logo” shows that the protein can
recognize a collectin of closely related DNA sequences and gives the preferred nucleotide pair at each position.
Recognition sequences can be ___ or ___ to the first exon
Proximal or distal
Regulatory proteins recognize and bind to bases in the ____ groove
major
The ____ groove presents a specific face for each of the specific base pairs
Major
A gene regulatory protein recognizes a
specific DNA sequence
The surface of the protein is extensively _____ to the surface of the DNA region to which it binds
complimentary
The gene regulatory protein makes a series of contacts with the DNA involving ____ possible configurations
4
What are the 4 possible configurations of base pairs for recognition of gene protein regulator
- possible hydrogen bond donor
- Possible hydrogen bond acceptor
- methyl groups
- Hydrogen atom
a typeical gene regulatory protein-DNA interaction involves _____ interactions
10-20
The parts of a DNA transcription factor: sequence specific transcription factors are _____
modular
What is the sequence of specific transcription factors Modules
N-terminus—DNA binding module— Dimerization module—Activation module– Regulatory module– C-terminus
Does every transcription factor have all the modules
no
What modules must a transcription factor have
DNA-binding module and Activation module
What is the function of the dimerization module
forms dimer with other protein subunits
What 2 modules can transcription factors have but don’t have to have
Dimerization module and Regulatory module
What is the function of the regulatory module
regulate transcription factor
DNA-binding domain structural motifs
- Helix-turn-helix
- Zinc finger motif
- Leucine zipper
- Helix-loop-helix
(also homeodomain and beta-sheet)
What is the simplest, most common DNA-binding motif
Helix-turn-helix
Explain helix-turn-helix
two alpha helices connected by short chain of amino acids that make the “turn” -turned at a fixed angle
The longer helix in a helix turn helix equals the
recognition module- DNA binding module-fits into major groove
Side changes of ____ in the helix turn helix recognize ____
Amino acids in the helix turn helix DNA motif
The helix turn helix proteins bind DNA as ____ in which the two copies of the recognition helix are separated by exactly one turn of the DNA helix
Dimers
The since finger proteins contain one or more ___ atoms as structural components
zinc
zinc finger proteins bind DNA in the ____ groove
major
Zinc finger domains found in tandem ____
clusters
What is the function of the zinc in zinc finger
stabilizes interactions with DNA
Leucine zipper motif has ____ DNA binding domains
-Two alpha helical DNA binding domains
Leucine zipper motif Dimerizes through ____ zipper region
leucine zipper region
the leucine zipper motif is named because of the way the
two alpha helices, one from each monomer, are joined together to form a short coiled-coil.
The leucine zipper proteins bind DNA as dimers where the two long alpha helices are held together by interactions between _______ amino acid chains
hydrophobic
The leucine zipper has a leucine residue every ___ amino acids down one side of the alpha helix in dimerization form: forms zipper structure
7
The dimerization of the leucine zipper grapes _____ like a clothespin
DNA
The activation domain overlaps _____ domain in a leucine zipper
dimer
The helix-loop-helix motif consists of a
short alpha helix connected by a loop to a second, longer alpha helix.
The helix-loop-helix motif can occur as ____ or _____
homodimers or heterodimers
what are the three domains or modules to the helix-loop-helix motif
DNA binding domain, dimerization domain, activation domain
Hereditary spherocytosis (HS) is an example of a ______ transcription factor mutation leading to disease
Zinc finger
__________ is a hemolytic anemia characterized by spherical and fragile red blood cells that lyse and release hemoglobin
Hereditary spherocytosis (HS)
What is the clinical presentation of Hereditary spherocytosis (HS)
hemolysis, anemia, splenomegaly
The HS clinical presentation ranges from ____ to ___ anemia and can be fatal
mild to severe
Hereditary spherocytosis (HS) is caused by mutations in genes for the erythrocyte membrane skeleton of _______
red blood cells- not making enough protein
_________ is the most common hereditary anemia in people of northern european descent (incidence 1/2,000)
Hereditary Spherocytosis (HS)
Hereditary Spherocytosis is _____ inherited
Dominantly
Hereditary Spherocytosis can be mutation in zinc finger protein gene _____
Klf1 (Kruppel-like factor 1)
______ zinc finger protein binds to promoters of all genes in EMS-and turns them on
KLF1
how many zing finger domains are there for KLF1
3
Klf1 has ___ exons
3, which encodes 3 zinc finger domains
A non-functioning KLF1 Zn finger protein leads to no ___
no EMS protein made and thus leads to HS (hereditary spherocytosis)
____ are critical amino acids for DNA binding in EMC proteins
RER
HS mutation: GAA to ____ or Glu to ___ in exon 3 (zinc finger domain 2)
GAT, Asp
_____ is the wild type DNA binding motif in exon 3 (zinc finger domain2) and the mutant form _____ leads to HS
- RER (Arg-Glu-Arg)
- RDR (Arg-Asp-Arg)
Normal KLF1 binds to DNA and
unwinds DNA causing transcription
HS KLF1 zinc finger binds to
opposite strand of DNA than the normal KLF1 so DNA cannot unwind and thus no transcription
Ways to identify Transcription factors
- Detection of sequence- specific DNA binding proteins
- Gel mobility shift assay
- EMSA: electrophoretic mobility shift assay
- use radioactive DNA from known promoter
- mix radioactive DNA fragment (regulatory DNA sequence) with protein extract from cell
- Run electrophoretic gel
- Proteins with DNA attached migrate according to size
- see shift of radioactive bad when protein is bound to DNA
- Isolate protein to identify
- affinity chromatography
- isolate DNA binding protein
- purification of sequence specific binding proteins
Technique that allows identification of the sites in the genome that a known regulatory protein binds to
CHIP: chromatin Immuno-precipitation
is Chromatin immunoprecipitation done on living cells
yes
DNA region involved in regulating and initiating transcription of a gene
Gene control region
Where transcription factors and RNA polymerase II assembles
Promoter
of the 25,000 human genes ___% (or _____ genes) encode gene regulatory proteins
8% or 2,000 genes
The gene control region contains the promoter and regulatory sequences to which
regulatory proteins bind to control rate of assembly process at the promoter
____ and ____ assemble at the promoter
RNA polymerase and general transcription factors
Gene regulatory proteins (activators or repressors) bind to regulatory sequences which can be
adjacent, far upstream or in introns downstream of the promoter
DNA looping and a mediator complex allow the gene regulatory proteins to interact with the proteins that assemble at the
promoter
The ____ serves as an intermediary between gene and regulatory proteins and RNA polymerase II
Mediator
Nucleosome remodeling and histone removal favors ____ by
transcription by increasing the accessibility of DNA to proteins
Histone acetylation makes it easier to ____ histones
remove
what are some of the different ways gene repressors inhibit transcription
- competitive DNA binding
- Masking activation surface (both proteins bind to DNA but the repressor binds to the activation domain of the activator protein)
- Direct interaction with the general transcription factors (the repressor binds to DNA and blocks assembly of general transcription factors)
- Recruitment of chromatin remodeling complexes (the repressor recruits a chromatin remodeling complex which returns the promoter to the pre transcriptional nucleosome state
- Recruitment of histone deacetylases (repressor attracts a histone deacetylase to the promoter-harder to remove deacetylated histones and open up DNA)
- Recruitment of histone methyl transferase (repressor attracts a histone methyl transferase which methylates histones.)(these methylated histones are bound to proteins which act to maintain chromatin in a transcriptionally silent form)
Gene regulatory proteins assemble into _____ on DNA
Complexes
Depending on the composition of complexes, proteins can be either ____ or ____
activating or repressing
The same protein can be part of an ____ or ___ complex
activating or repressing complex
What is meant by regulation by committee
Depending on the composition of complexes, proteins can be either activating or repressing and the same protein can be part of an activating or repressing complex
How are gene regulatory proteins controlled
- Synthesis
- Ligan binding
- Covalent modification phosphorylation
- Addition of subunit
- Unmasking
- Nuclear entry
- Proteolysis
What are the alpha like chains
zeta and alpha
what are the beta like chains
epsilon, gamma, delta, beta
Embryonic Hb=
zeta and epsilon
fetal Hb=
Alpha and gamma
Adult Hb=
Alpha and beta
Globin genes are arranged in ____ fashion
linear
Globin genes are ordered in the ___ to ___ direction in the same sequence of activation and expression during embryonic, fetal, and adult development
5’ to 3’
Globin genes are on chromosomes
16 (zeta, alpha) and #11 (epsilon, gamma, delta, beta)
The beta globin gene regulation is a ____kb region containing ____ beta globin genes and locus control region (LCR
100 kb, five
Regulatory proteins for globin genes bind to the ____
LCR (locus control region)
Understanding of globin gene regulation may allow the induction of ______ in sickle cell anemia
fetal hemoglobin
What is the beta globin gene regulation order
LCR, Epsilon, gamma(G), Gamma(A), delta, beta
