Lecture Notes Regulating Gene Expression Flashcards
How is gene expression regulated in prokaryotes?
Prokaryotes make certain proteins only when they are needed
To shut off the supply of a protein, the cell can
1) downregulate mRNA transcription (most effective)
2) hydrolyze mRNA, preventing translation
3) prevent mRNA translation at the ribosome
4) hydrolyze the protein after it is made
5) inhibit the proteins function
Repressor protein bound at site where RNA wants to bind and prevents initiation of transcription
Negative regulation
Activator protein enhances transcription
Positive regulation
Regulating gene transcription allows E. coli to (blank) in an ever changing environment
Conserve energy
(Blank) is the easiest sugar to metabolize
Glucose
Lactose is (blank)
B-galactoside
3 proteins needed for the uptake and metabolism of lactose
B-galactoside permease
B-galactosidase
B-galactoside transacetylase
Carrier protein that moves lactose into the cell
B-Galactoside permease
Hydrolyses lactose
B-galactosidase
Transfers acetyl groups from acetyl coA to certain b galac
B-galactoside transacetylase
(Blank) stimulates expression of B-Galactosidase
Lactose
Lactose is a (blank)
Inducer
Lactose increases or decreases mRNA
Increases
E. coli makes (blank) first before proteins
MRNA
Compounds that stimulate protein synthesis are called
Inducers
(Blank) are made at a constant rate
Constitutive proteins
2 ways to regulate metabolic pathways
- Regulation of enzyme activity
- regulation of enzyme concentration
End product feeds back, inhibiting the activity of enzyme 1 only, quickly blocking pathway
Regulation of enzyme activity
End product blocks the transcription of all 5 genes- no enzymes produced
Regulation of enzyme concentration
Gene cluster with a single promoter
Operon
A typical operon consists of:
A promoter
Two or more structural genes (z, y, and a)
An operator
A short sequence between the promoter and the structural genes that binds regulatory proteins
Operator
Three ways to control operon transcription
1) an inducible operon regulated by a repressor protein
2) a repressible operon regulated by an activator protein
3) an operon regulated by an activator protein
Repressor binds operator sequence and blocks RNA poly from binding- no requirement and genes transcribe
Inducible system- lactose absent
Repressor protein has binding sequence for lactose - RNA polymerase binds to breakdown and metabolize lactose
Inducible system: lactose present
Trp operon is a (blank) system
Repressible
Trp operon- amino acid
Incorporated into proteins
The trp repressor binds the operator, and RNA synthesis is blocked
Tryptophan present
Repressor dissociates from the operator, and RNA synthesis proceeds
Absence of tryptophan
Metabolic substrate is a
Inducer
Regulatory protein is a
Repressor
Metabolic substrate interacts with a regulatory protein- repressor can’t bind to operator and transcription proceeds
Inducible system
Control catabolic breakdown pathways
Inducible systems
Turned on when substrate is available
Catabolic pathways
Metabolic product is a
Co-repressor
A metabolic product binds to a regulatory protein, which then binds to the operator and blocks transcription
Repressible systems
Control anabolic (build) pathways
Repressible systems
Turned on until product concentration becomes excessive
Repressible systems
E. coli can use (blank) to increase transcription
Positive control
If glucose and lactose levels are both high, the (blank) is not transcribed efficiently
Lac operon
Efficient transcription requires (blank) to increase transcription
Binding of an activator protein to lac operon promoter
Example of activator protein
CAMP bund to CRP
Low glucose means CRP
Bound to promoter
High glucose means CRP
Not bound
A system or gene regulation in which presence of a preferred energy source represses other catabolic (break-down) pathways
Catabolite repression
RNA polymerases bind and are orientated at promoters so that (blank)
The correct DNA strand is transcribed
All promoters have (blank) that allow them to be recognized by RNA polymerase
Consensus sequences
Different classes of consensus sequences are recognized by regulatory proteins called
Sigma factors
Bind to RNA polymerase and direct it to certain promoters
Sigma factors
Genes for proteins with related functions may be at different locations in the genome, but share consensus sequences and can be recognized by
Sigma factors
Is active most of the time and binds to consensus sequences of housekeeping genes
Sigma factor 70
Genes normally expressed in actively growing cells
Housekeeping genes
How is gene expression regulated in eukaryotes?
1) remodeling chromatin- epigenetics
2) transcriptional control
3) processing control
4) transport control
5) mRNA stability control
6) translational control of protein synthesis
7) posttranslational control of protein activity
8) protein degradation
Regulation- prokaryotic vs eukaryotic - BOTH
Use DNA protein interactions and negative/positive control to regulate gene expression
Beginning steps in initiation of eukaryotic transcription
TATA box in promoter bound by TFIID
Transcription initiation complex includes
TFIID- TFIIB- TFIIF- TFIIE- TFIIH
Binds to TATA box
TFIID
Binds both RNA polymerase and TFIID, and helps identify the transcription initiation site
TFIIB
Prevents nonspecific binding of the complex to DNA and helps recruit RNA polymerase to the complex
TFIIF
Similar to function of bacterial sigma factor
TFIIF
Binds to the promoter and stabilizes the denaturation of the DNA
TFIIE
Opens up the DNA for transcription
TFIIH
Transcription factor specificity does what
Plays an important role in cell differentiation
Regulatory sequences that bind transcription factors that activate transcription or increase rate of transcription
Enhancers
Bind transcription factors that repress transcription
Silencers
Structural motifs mediate (blank) which means what
DNA binding
Fundamental to differentiation
What is a common structural motif?
Helix-turn-helix
For DNA recognition the structural motif must:
- fit into a major or minor groove
- have amino acids that can project with interior of double helix
- have amino acids that can bond with interior bases
What does the lac repressor do and what is it often called?
Bonds DNA and inhibits ability of TF binding
Dimer
Expression of transcription factors underlies (blank)
Cell differentiation
During development, cell differentiation is often mediated by
Changes in gene expression
All differentiated cells contain (blank)
Entire genome
The expression of just 3 TF is sufficient to transform (blank)
Fibroblast into a neuron
Coordinating gene expression
- separate genes unlike prokaryotes
- same sequence of DNA in front of multiple genes
Process in which a multicellular organism undergoes a series of progressive changes that characterizes its life cycle
Development
Sets the fate of the cell
Determinaron
Determination
Sets the fate of the cell
The process by which different types of cells arise
Differentiation
Differentiation
The process by which different types of cells arise
Morphogenesis
Organization and spatial distribution of differentiated cells
Organization and spatial distribution of differentiated cells
Morphogenesis
Increase in body size by cell division and cell expansion
Growth
Growth
Increase in body size by cell division and cell expansion
Why do determination and differentiation occur?
Differential gene expression
Morphogenesis involves differential gene expression and the interplay of signals between cells:
- cell division
- cell expansion in plants
- cell movements are important in animals
Apoptosis is essential in
Organ development
Growth occurs by
Increasing the # of cells or enlargement of existing cells
Cell fates become progressively (blank) during development
More restricted
Cell fate determination is influenced by
Gene expression and the extracellular environment
Is determination before or after differentiation?
Determination
Changes in biochemistry, structure, and function that result in (blank)
Different cell types
Potential to differentiate into other cell types
Cell potency
Can differentiate to any cell type
Totipotent
Can develop into most cell types, but cannot form new embryos
Pluripotent
Can differentiate into several related cell types
Multipotent
Can produce only one cell type- their own
Unipotent
How does one egg cell produce so many different cell types?
2 processes for cell determination
- cytoplasmic segregation
- induction
Cytoplasmic segregation
Unequal cytokinesis
Induction
Cell to cell communication
Cytoplasmic segregation can determinate (blank)
Polarity and cell fate
Factors within a zygote or egg are not distributed evenly and end up in different daughter cells after division
Cytoplasmic segregation
Developing a “top” and “bottom”- can develop very early; yolk and other factors are distributed asymmetrically
Polarity
Top-nothing and bottom-small sea urchin = (blank) cut
Horizontal
Top and bottom- small sea urchins = (blank) cut
Vertical
The cytoskeleton contributes to
Asymmetric distribution of cytoplasmic determinants
Microtubules and microfilaments have (blank)
Polarity
Cytoskeletal elements can bind motor proteins that (blank)
Transport the cytoplasmic determinants
Communication from one cell to another can (blank)
Determine cell fates
Cells in a developing embryo influence one another’s developmental fate via chemical signals and signal transduction mechanisms
Induction
What is the role of gene expression in development?
- all cells in an organism have the same genes, but each cell expresses only certain ones
- the mechanisms that control gene expression during cell fate determination and differentiation
Inducers mediate signal transduction to (blank)
Dictate differential gene expression
Explain how inducers mediate signal transduction to dictate differential gene expression
- inducer molecules bound by receptors on surface of cell
- bound receptor creates a signaling cascade internally that sends TFs into nucleus to cause gene expression and bind DNA
B cell development is directed by
Signaling that causes differential gene expression
Inducer molecule signal mediates the expression of different genes which causes the cell to (blank)
Survive, perliforate, and proceed in development
2 things that happen after the receptor is bound
1) conformational change = receptor shape inside cell which changes to signal and is bound to Fit3 (chemical inducer)
2) phosphorylation
Differential gene transcription is a (blank)
Hallmark of cell differentiation
Process of transcription and differentiation in the formation of muscle cells
Mesoderm cells- myoblasts- muscle cell
-event blocks behavior of cell when dividing in order to push differentiation forward
Differential gene expression drives (blank)
Development
How is gene expression linked to the way we look?
Pattern formation and morphogenesis
Creation of body form
Morphogenesis
The process that results in the spatial organization of tissues and organisms
Pattern formation
Morphogenesis involves
Cell division and differentiation, apoptosis
Pathways for apoptosis in C elegans
CED-9 to CED4 to CED-3 to apoptosis
Pathways for apoptosis in human neuron
BcL-2 to Apaf-1 to caspase-9 to caspase-3 to apoptosis
Dictate what differentiated cells become
Organ identity genes
The four organs in a flower are determined by the four groups of cells in the (blank)
Meristem
A protein called LEAFY controls (blank)
Transcription of organ identity genes
Plants with loss of function mutations of LEAFY (blank)
Do not produce flowers
Transgenic orange trees, expressing the LEAFY gene coupled to a strongly expressed promoter, (blank)
Flower and fruit years earlier than normal trees
Morphogen gradients provide (blank)
Positional information
The position of each cell is defined by (blank)
Concentration of morphogen
Describe example of specification of the vertebrate limb
Higher shh signaling drives differentiation of little finger vs thumb
What was discovered when scientists studied morphogens in fruit flies?
The head, thorax, and abdomen are each made of several fused segments and different body parts arise from these different segments
In fruit flies, when do segments appear?
early in development
In fruit flies, by the early larval stage what has already occurred?
cell fates already determined
When the embryo of fruit flies is first formed, what happens?
In the 1st 12 mitotic divisions, there is no cytokinesis, forming a multinucleate embyro and morphogens can diffuse easily in the embryo.
Steps of cell determination were studied using experimental genetics
- developmental mutations were identified
- mutants were compared with wild types to identify genes and proteins
- experiments confirmed gene and protein functions
Three gene classes of determination:
- Maternal effect genes
- Segmentation
- Hox
Set up the major axes of the egg
Maternal effect genes
Maternal effect genes
set up the major axes of the egg
Segmentation genes
determine boundaries and polarity of each segment
Determine boundaries and polarity of each segment
Segmentation genes
Determine which organ will be made at a given location
Hox genes
Hox genes
determine which organ will be made at a given location
transcribed in cells of the mother’s ovary; the mRNAs are passed to the egg
maternal effect genes
(blank) and (blank) are genes that help determine the anterior-posterior axis of the embryo
Bicoid and nanos
subject to unequal distribution
maternal effect genes
What establishes the hunchback gradient?
Bicoid and nanos genes
Nanos (blank)
inhibits
Bicoid (blank)
stimulates
Mutations result in posterior structures being replaced by reversed anterior structures
Segmentation genes
3 classes of segmentation genes
gap, pair rule, segment polarity
organize broad areas; mutations result in omission of several body segments
Gap genes
divide embryo into units of two segments each; mutations result in every other segment missing
Pair rule genes
determine boundaries and anterior-posterior organization in individual segments
Segment polarity genes
Particular Hox gene encodes for
particular organ/body part
encode transcription factors that are expressed in different combinations along the length of the embryo
Hox genes
What determines cell fate in each segment of organism?
Hox genes
In Drosophilia, Hox genes determine (blank)
Segment identity
Hox genes have a 180 base pair sequence called the (blank)
Homeobox
The homebox encodes a 60 amino acid sequence called the (blank)
Homeodomain
The homeodomain binds to
specific DNA sequences in the promoters of target genes
Is differentiation reversible?
yes
A zygote is totipotent meaning it can
give rise to every cell type in organism
As development proceeds, cells become determined and lose their (blank)
totipotency
What is an example in which differentiated plant cells can be turned totipotent?
Carrot cloning
Nuclear transfer experiments show that genetic material from a single cell can be used to (blank) animals
clone
Initial cloning experiments show reversible nature of (blank)
differentiation
Cloning experiments indicated that
-no genetic info is lost as cell passes through developmental stages
Step 1 to cloning a mammal (Dolly)
took mammary epithelial cells and cultured them (#1 sheep)
Step 2 to cloning a mammal (Dolly)
harvested eggs from #2 sheet and eneucleated them
Step 3 to cloning a mammal (Dolly)
fused 1 mammary epithelial cell with 1 eneucleated egg
Step 4 to cloning a mammal (Dolly)
induced cells to divide- embryos
Step 5 to cloning a mammal (Dolly)
embryos implanted into 3rd sheep
What did the cloning of Dolly the sheep show?
fully differentiated cell from a mature animal can revert to totipotent
What are the benefits of cloning animals?
- increase # of valuable animals (ex- trangenic animals with genes with therapeutic properties)
- preservation of endangered species
- preservation of pets
What is one specific example involving a positive benefit from cloning an animal?
cow genetically engineered to make HGH in milk cloned to produce hormone for children with growth hormone deficiency
Rapidly dividing, undifferentiated cells that can differentiate into several cell types
Stem cells
Stem cells
rapidly dividing, undifferentiated cells that can differentiate into several cell types
In plants, stem cells are in the (blank)
meristems
In mammals, stem cells occur in tissues that require frequent (blank)
replacement (skin, blood, intestinal lining)
Two types of adult stem cells
Hematapoetic and mesencymal stem cells
Stem cell transplantation often occurs
after cancer
stem cell “healing” therapy
stem cells may be able to insert into the tissue and differentiate or induce tissue regeneration
Experiments show that damaged tissues can heal more effectively if (blank)
stem cells are injected into tissue
Using ESCs to obtain (blank)
pluripotent stem cells
ESCs (blank)
repair tissue, recover from disease, heart attack
ESCs can be (blank)
harvested from human embryos conceivied by in-vitro
2 problems with harvesting ESCs from embyros
- some people object to destruction of human embryos
- stem cells could provoke an immune response in a recipient