Meeting 12 - gel shifts/lac operon Flashcards
What is a gel shift?
An assay which we use to measure sequence-specific binding by transcription factors
Homodimer
a protein composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues
Heterodimer
a protein composed of two polypeptide chains differing in composition in the order, number, or kind of their amino acid residues
What do Transcription facotrs usually bind as?
homodimers
What are oligonucleotides?
Short sequences of DNA, perhaps 15 to 20 base pairs that are very similar to primers that we get for PCR
Native gels
Native - (non-denaturing) - Separate proteins according to the net charge, size and shape of their native structure
how to check the specificity of protein-DNA interaction on gels (way#1)
add some nonspecific DNA
- if the band is lost, we know it is nonspecific
- if the band in maintained we know its specific
how to check the specificity of protein-DNA interaction on gels (way#2)
add more unlabeled DNA, and add DNA with mutations. run gel and observe the shifts that take place
how to check the specificity of protein-DNA interaction on gels (way#3)
add antibodies specific for transcription interactions.
-If transcription factor is combined to the antibody with 1 domain and the DNA sequence with another domain, you will see a super shift
-If the antibody is directed against the DNA-binding domain, then it might actually inhibit binding of the transcription factor to the piece of DNA (lose DNA binding completely)
why does e.coli prefer glucose over lactose?
it’s less complex - ready to metabolize for energy.
lactose needs to use energy to make glucose to later get energy
cis
An element is considered to be cis-acting if it can only affect other elements on the same DNA strand it is on.
trans
Elements that are trans-acting are able to affect DNA strands they are not a part of.
what do you need for a gel shift
- non-specific DNA
- transcription factors
- double-stranded, radio labeled DNA
what is allolactose
natural inducer of the lac operon
how does the lac operon use allolcatose (inducer)
Allolactose binds to the lacl protein (repressor), this induces a conformation change in lacl, it dissociates from the lac operator, so that RNA polymerase can transcribe the lacz gene, and the cell can use lactose as an energy source.
