Control Of Gene Expression 1 Flashcards
What makes cells different?
Structure and function
It was previously thought that cells lose some genes so only certain proteins are then expressed. However, if normal skin cells are isolated from a frog and the nucleus is implanted into a denucleated cell a normal embryo forms. This obviously shows that NO genes are lost.
Different cells all contain the same set of genes but express different sets of proteins. Cell differentiation of cells depends on gene expression.
Different cell types -diff proteins
1-4
Control of Gene Expression (6)
1) transcriptional control
2) RNA processing control
3) RNA transports and localization control
4) translation control
5) mRNA degradation control
6) protein activity control
Gene regulation requires
1) short stretches of DNA of defined sequence - recognition sites for DNA binding proteins
2) Gene regulatory proteins - transcription factors that will bind and activate gene
Steps of DNA Motif Recognition
- association of regulatory proteins with major groove
- proteins recognize and bind to bases in major groove
- major groove presents a specific face for each of the specific base pairs
- gene regulatory protein recognizes a specific sequence of DNA
- surface of protein is complementary to surface of DNA region to which it binds
- series of contacts is made with the DNA involving 4 possible configurations
Four Base Pair Configurations in Motif Recognition
Blue- possible h bond donors
Red- possible h bond acceptors
Yellow- methyl group
White- h atom
What are the mandatory parts of a DNA transcription factor? What are the non mandatory parts?
Every factor will have a DNA-Binding Molecule.
Dimerization module- form diners with other subunits
Activation module- turn on gene
Regulatory module- regulate transcription factor
Evidence for trans factors being modular
Experiment
What are the 4 kinds of DNA-binding domain structural motifs?
1) Helix-turn-Helix
2) Zinc Finger motif
3) Leucine Zipper
4) Helix-loop-Helix
(Also Homeodomain and Beta-sheet)
Helix-Turn-Helix
- Simplest most common motif
- two alpha helices connected by short chain of amino acids that make the turn (turn is at fixed angle)
- longer helix = recognition module - DNA binding module - fits into major groove
- side chains of amino acids recognize DNA motif
- symmetric diners: ind DNA as dimers
Zinc Finger Domain
- Includes Zinc atom
- when drawn out, the amino acid sequence looks like a finger projection
- 3D structure does not look like finger projection does not
- Binds to major groove of DNA
- Found in tandem clusters
- Stabilizes interactions with DNA
- Multiple contact points
Leucine Zipper Motifs
- Two alpha helical DNA binding domains
- Grabs DNA like clothespin
- Activation domain overlaps dimer domain
- Interactions between hydrophobic amino acid side chains (leucine)
- Dimerizes through leucine zipper region (homo-/hetero-)
- One leucine residue every 7 AA down on side of alpha helix in domain forms the zipper structure
Helix-Loop-Helix Domain
-Consists of a short alpha chain connected by a loop to a second longer alpha chain
-can occur as homodimers or heterodimers
Three domains or modules to this protein: DNA binding domain, dimerization domain, activation domain
Homeodomain Proteins
- Contain Homeodomain which consists of 3 alpha helices
- helix-turn-helix motif
Beta Sheet DNA Recognition Proteins
- 2 stranded beta sheet
- Beta sheet consists of beta strands
- connected laterally by two or three backbone h bonds
- forms twisted, pleated sheet
- binds to major groove of DNA
What disease is an example of Zn finger transcription factor mutation?
Hereditary spherocytosis
What are the clinical presentations of HS?
Hemolysis, anemia, splenomegaly
Symptoms range from mild to severe, eg- fatal anemia
HS is caused by mutation in genes for the ____ ___ ___ of _BCs - not making enough protein.
Erythrocytes membrane skeleton
Red
HS is ____ inherited.
The purpose of the EMS is:
Dominantly
-Conferring durability and stability to RBCs while they pass through tight capillary spaces and make 1/2 million passages in circulation.
KLF1 Zinc Finger Protein
(Kruppel-like factor 1)
Mutation leads to…
Binds to promoters of all genes in EMS - turns them on
Non-functioning KLF1 Zn finger protein, no EMS made, leads to HS
In HS, what is mutated and what is the result?
GAA —> GAT or Glu —> Asp or RER —> RDR
Domain 2 is mutated
No transcription occurs
Defective: makes less RNA from target promoters of EMS genes therefore less protein is available and HS develops
Describe the identification of transcription factors using radioactive DNA.
- 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 band when protein is bound DNA
- isolate the protein to identify it
Allows Detection of sequence-specific DNA binding proteins
- Gel Mobility shift assay
- EMSA: electrophoretic mobility shift assay
Describe the identification of transcription factors using affinity chromatography
Isolate the DNA binding protein
Purify the sequence specific binding proteins
Start Broad- identify any DNA binding protein, then Get Specific- use only 1 promoter recognition sequence
Identification of a DNA binding sequence
CHIP: Chromatin Immuno-Precipitation
- technique allows identification of the sites in the genome that a known regulatory protein binds to
- done in living cells
- PCR product at end can be used to identify sequence
Gene Control Region
- DNA region involved in regulating and initiating transcription of a gene
- Includes the promoter (where transcription factors and RNA polymerase II assembles)
- Regulatory sequences to which regulatory proteins bind to control rate of assembly process at promoter
- 9% of human genes encode reg proteins
- control of mammalian genes is extremely complex
- few generalities can be made
Gene Control Region
___ Polymerase and ___ ____ factors assemble at the promoter.
Other gene regulatory proteins (___ or ___) bind to regulatory sequences which can be adjacent, far upstream or introns downstream of the promoter.
RNA
General Transcription
Repressors or activators
Transcriptional Activation Model
DNA loops upon itself and a mediator allows the gene reg proteins to interact with the proteins assembled at the promoter
The mediator serves as an intermediate between gene reg proteins and RNA polymerase II
Nucleoside remodeling and histone removal favors transcription by increasing accessibility of ____ to ____.
DNA to proteins
What are the 4 ways Proteins modify DNA (local chromatin structure)
Nucleosome remodeling
Nucleosome removal
Histone Replacement
Histone modification (eg- acetylation makes it easier to remove histones)
True or False: In competitive DNA binding activators and repressors have separate binding sites.
False, they compete for the same binding site
In the masking form of Gene repression which protein binds to the DNA? How does this block activation?
Both proteins (activator and repressor) bind to DNA but the repressor binds to the activation domain of the activator protein, stopping activation.
When direct interaction with the general transcription factors occurs…
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 transcriptional nucleosome state
Recruitment of Histone deacetylases
The repressor attracts a histone deacetylase to the promoter - Harper to remove deacetylated histones and open up DNA
Recruitment of histone methyl transferase
- the repressor attracts a histone methyl transferase which methylated histones
- these methylated histones are bound to proteins which act to maintain chromatin in a transcriptionally silent form
Gene regulatory proteins assemble into complexes on DNA
In solution the proteins are separate floating. On DNA they assemble into complexes
- depending on the composition of the complexes, proteins can be activating or repressing.
- the same protein can be apart of an activating or repressing complex
- regulation by committee (group effort)
Name the 7 control processes of Gene reg proteins
A. Synthesis B. Ligand binding C. Covalent modification-phosphorylation D. Addition of a subunit E. Unmasking F. Nuclear Entry G. Proteolysis
What are the alpha-globin chains
Zeta and alpha
What are the beta-globin like chains
Epsilon, gamma, delta, beta
Hemoglobin Switching
- 2 alpha type glob is and 2 beta type glob is
- embryonic = zeta, epsilon
- fetal = alpha, gamma
- adult = alpha, beta
- embryonic to fetal to adult (switching)
- not clear how this occurs
Globin Gene Expression
Beta-globin genes are arranged in linear fashion, in 5’ to 3’ direction in all stages of development (emb, fetal, adult)
Beta Globin Gene Regulation
100 kB region containing five beta globin genes and locus control region (LCR)
Far upstream in sequence but required for transcription
Regulatory proteins bind to LCR
Sickle cell disease - sickle beta globin - serious anemia
(This is a cure)
If make fetal Hb, not much needed to cure sickle cell
-switch back from adult to fetal Hb is cure. Understanding of globin gene reg may allow the induction of fetal hemoglobin in sickle cell anemia cure
