Lecture 11 - Regulation of Gene Expression Flashcards
What are 3 pieces of evidence that prove that all cells contain the same genetic material?
- The nucleus of a skin cell from an adult frog transplanted into an enucleated egg can give rise to an entire tadpole.
- In many types of plants, differentiated cells retain the ability to “dedifferentiate,” so that a single cell can form a clone of progeny cells that later give rise to an entire plant.
- A differentiated cell nucleus from an adult cow introduced into an enucleated egg from a different cow can give rise to a calf.
What are the 6 stages at which eukaryotic gene expression can be controlled?
- Transcription
- RNA processing
- RNA transport and localization
- Translation
- mRNA degradation
- Protein activity
What is the simplest, most common DNA binding motif? Explain how it binds DNA.
Helix-turn-helix:
- The C-terminal alpha helix = recognition helix because it participates in sequence-specific recognition of DNA
- The N-terminal alpha helix functions primarily as a structural component that helps to position the recognition helix
Where does the recognition helix fit in DNA?
R groups interact with bases at the DNA major groove
What are super secondary structures? Eg?
Common sets or combinations of secondary structures that have distinct functions
Eg: helix-turn-helix to bind DNA
How do a lot of helix-turn-helix binding proteins bind DNA?
As dimers in which the two copies of the recognition helix are separated by exactly one turn of the DNA helix and bind a palindromic sequence
What are 3 different motifs of DNA binding protein super secondary structures?
- Helix-turn-helix
- Zinc finger
- Leucine zipper
What are zinc finger DNA-binding motifs common for?
Hormone receptor binding to DNA
Describe the zinc finger DNA binding motif.
Zinc chelates 2 histidines and 2 cysteines that forms a “finger” that is capable of interacting with the major groove of DNA
How do a lot of zinc finger binding proteins bind DNA?
As dimers in which the two copies of the recognition sequence are separated by exactly one turn of the DNA helix and bind a palindromic sequence
Describe the leucine zipper DNA binding motif.
Leucine at every 7th AA: 2 identical amphiphatic alpha helical DNA- binding domains dimerize through their alpha-helical leucine zipper region (top) to form an inverted Y-shaped structure. Each arm of the Y is formed by a single alpha helix, one from each monomer, that mediates binding to a specific DNA sequence in the major groove of DNA. Each a helix binds to one-half of a symmetric DNA structure: palindrome.
What can be determined if each 7th AA is the same in an alpha helix?
There are 3.5 AAs per turn of the alpha helix, so this would mean that the alpha helix is amphiphatic, meaning one side is hydrophilic and the other is hydrophobic
How do DNA binding proteins recognize DNA sequences?
The proteins decode the functional groups that are accessible on the side of the bases as revealed in the major grooves of DNA:
- H bond acceptors
- H bond donors
- H atoms
- Methyl groups
What is the purpose of heterodimerization of leucine zipper proteins?
Heterodimerization of leucine zipper proteins can alter their DNA-binding specificity: the two different monomers can combine to form a heterodimer, which now recognizes a hybrid DNA sequence, composed 2 non-palindromic sequences
Why can’t DNA binding proteins bind at the DNA minor grooves?
Because the side patterns are not unique to particular base pairs
How many contacts does a protein-DNA interface usually have? What are these?
20
H bonds
What must be true of DNA-protein contacts?
Must be close enough to exclude water or else the H bonds would be broken
Is it possible to predict what DNA sequence a protein will bind to based on the primary structure and the super secondary structure motif?
NOPE
What is a very common DNA-AA binding contact?
Guanine-Arginine
What are restriction enzymes? What do they allow us to do?
DNA binding proteins that contain endonuclease activities and recognize specific DNA sequences
Engineer DNA and recombine sequences
What is an operon? What does this allow for?
Cluster of genes transcribed as a single mRNA molecule
Allows their expression to be controlled coordinately
What is a common bacterial operon? What does it code for?
trp = cluster of enzymes needed for tryptophan biosynthesis
Do eukaryotes have operons?
NOPE
Describe how the trp operon is regulated.
In absence of Trp, repressor does not bind the operator sequence on DNA
In presence of Trp, Trp binds the repressor which activates it to bind the operator sequence, thereby blocking RNA polymerase binding to DNA and mRNA synthesis
Where is the operator sequence located on prokaryotic DNA?
The promoter region
How does the binding of Trp affect the repressor protein in prokaryotes? What is this called?
It changes its conformation allowing it to bind DNA: binding increases the distance between the two recognition helices in the homodimer, allowing the repressor to fit snugly on the operator
= NEGATIVE CONTROL
How do we regulate genes?
Combination of positive and negative regulation by transcriptional repressors and transcriptional activators
Can bacterial gene regulatory proteins act as both transcriptional activators AND repressors? How?
Yes depending on the placement of their DNA-binding sites aka where it binds on different genes
Describe the regulation of the Lac operon.
LacZ, the first gene of the Lac operon, encodes the enzyme b-galactosidase, which breaks down the disaccharide lactose to galactose and glucose. Keep in mind prokaryotes will favor glucose metabolism so will only want to express this gene when lactose is present and glucose is not.
- Lactose addition increases the concentration of allolactose, an isomer of lactose, which binds to the repressor protein and removes it from the DNA.
- Glucose addition decreases the concentration of cyclic AMP; because cyclic AMP no longer binds to CAP, this gene activator protein dissociates from the DNA, turning off the operon.
(many other repressor sites than this one though)
Is the expression Lac operon ever completely shut down?
NOPE, a small amount of the enzyme b-galactosidase is required to convert lactose to allolactose, thereby permitting the Lac repressor to be inactivated when lactose is added to the growth medium
Lac operon:
+ glucose
+ lactose
Operon ON or OFF? Why?
OFF
Because CAP not bound
Lac operon:
+ glucose
- lactose
Operon ON or OFF? Why?
OFF
Because Lac repressor bound AND CAP not bound
Lac operon:
- glucose
- lactose
Operon ON or OFF? Why?
OFF
Because Lac repressor bound
Lac operon:
- glucose
+ lactose
Operon ON or OFF? Why?
ON
Because CAP bound and Lac repressor unbound
What are the 2 functional domains of transcription factors? How many of each?
- DNA-binding domains: 0 or 1
2. Transactivation domains: 1 or more
What is another name for transcription factors?
Gene activator proteins
What are 2 other names for the transactivation domain of transcription factors?
- Protein-protein interaction domain
2. Transcription activation domain
Do transcription factors need to be able to bind to DNA to be able to regulate gene expression?
NOPE
Describe the modular structure of a transcription factor.
Some transcription factors have independent DNA-binding and transactivation domains and the DNA binding domain positions the transcription factor specifically so that the transactivation domains can activate a specific gene
What are the 3 DNA elements that regulate gene expression?
- Promoters
- Enhancers
- Repressors
What is the function of promoters?
Establish minimum conditions for baseline transcriptional activity
What is the role of enhancers?
Regulate transcriptional activity
Do the enhancers contain a promoter function? What does this mean?
NOPE
Unable to initiate transcription without a promoter
Can the enhancers be outside of the promoter region? What does this mean?
YUP
They are position independent
Describe the enhancer sequences. What does this mean?
Palindromes = orientation independent and commonly bound by dimers
What are the 2 types of transcriptional factors that bind to enhancers?
- Constitutive transcription factors
2. Inducible transcription factors
What are the 3 types of inducible transcription factors?
- Second-messenger dependent
- Hormone receptors
- Tissue-specific
When are constitutive transcriptional factors expressed? What genes do they regulate?
Expressed at all times and regulate the expression of normal function/housekeeping genes
When are inducible transcriptional factors expressed?
Turned on and off in response to specific signals
What is an example of a second messenger dependent inducible transcription factor? How does it work?
CRE = cAMP response element
Phosphorylated in presence cAMP so it binds to a particular palindromic DNA sequence
What % of proteins encoded in our genome encode gene regulatory proteins?
8%
Where can DNA regulatory sequences be located? 4 options
- Adjacent to the promoter
- Far upstream of the promoter
- Within introns
- Downstream of the gene
What does DNA looping allow?
DNA looping allows gene regulatory proteins bound at any DNA positions to interact with the proteins that assemble at the promoter, like the Mediator
What are the 4 ways in which eukaryotic transcription factors can direct local alterations in chromatin structure to stimulate transcription initiation?
- Remodeled nucleosomes by chromatin remodeling complexes
- Histone removal with histone chaperones
- Histone replacement with histone chaperones
- Specific pattern of histone modification by histone modifying enzyme
How do specific patterns of histone modification by histone modifying enzymes work?
Usually make histones resistant to unpackaging
What is the usual role of histone acetylation?
Makes it easier for histone chaperones to remove them from nucleosomes
How do transcription factors work together? How/When is this observed?
They work synergistically or competitively and have additive effects
This is typically observed between different gene activator proteins from the same organism and even between activator proteins from different eucaryotic species when they are experimentally introduced into the same cell
What are the 6 ways in which eukaryotic gene repressor proteins can operate?
- Compete for binding with activator proteins to the same regulatory DNA sequence
- Masking the activation domain surface of the activator factor (activator can still bind to DNA)
- Direct interaction with the general transcription factors to block their assembly
- Recruitment of chromatin remodeling complexes which returns the nucleosomal state of the promoter region to its pre-transcriptional form
- Recruitment of histone deacetylases thereby stabilizing the histones to maintain the chromatin in a transcriptionally silent form
- Recruitment of histone methyl transferases to methylate histones so that proteins can bind to them and maintain the chromatin in a transcriptionally silent form
Does the repressor factor need to be bound to DNA to mask the activation domain surface of the activator factor?
NOPE
What is an example of a repressor protein inhibiting transcription by direct interaction with the transcription factors?
Preventing release of RNA pol from the initiation factors
What are architectural proteins?
Main role is to bend the DNA to allow the cooperative assembly of the transcription factors
What is another name for architectural proteins?
DNA-bending proteins
How do multiple sets of gene regulatory proteins work together to influence transcription initiation at a promoter?
They work together to increase or decrease the probability of initiating transcription
What are insulators?
DNA sequences that directionally block the action of enhancers
What are barrier sequences?
DNA sequences that prevent the unpacking of heterochromatin by stabilizing it
What super secondary structure does the repressor use to bind the bacterial Trp operon?
Helix turn helix
