Flashcards in Lecture 11 - Regulation of Gene Expression Deck (66)
What are 3 pieces of evidence that prove that all cells contain the same genetic material?
1. The nucleus of a skin cell from an adult frog transplanted into an enucleated egg can give rise to an entire tadpole.
2. 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.
3. 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?
2. RNA processing
3. RNA transport and localization
5. mRNA degradation
6. Protein activity
What is the simplest, most common DNA binding motif? Explain how it binds DNA.
1. The C-terminal alpha helix = recognition helix because it participates in sequence-specific recognition of DNA
2. 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?
2. Zinc finger
3. 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?
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?
What is a very common DNA-AA binding contact?
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?
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)