How large is the bubble?
Load # of polymerases,nick the strand to relieve the strain, then ligate nick back.
We get unwound molecule. We can measure amount of supercoil (it will migrate faster in the
gel than relaxed)
Shows how big the bubble is Shows how big the bubble is
How polymeraseclears to elongation
We examine three different ways from initiation to elongation (hypotheses):
Inchworming - stretches then tail catches up
Scrunching - proved to be true (polymerasedoesn’t change)
Fret alex - we are not going to make a mistake since that alone will decrease energy
transfer and it will confound results Inhibits first few bases
SDS linearized the amino acid chain and coverit to have equally negative charge - however,if you
take SDS, the protein is not likely to get back the original configuration. It not easy to form the 3D
structure of protein.
Urea is less harsh and will retain the structure of protein (i.e. Rnase is very robust, it retains its
There are different possible combinationsof reconstitution(rifampacin-resistant at the right). As
long as you have all the subunits.
Good experiment - tells where is the rifampacin resistant is - in the beta Polymerasegoes to the active site
Radiolabel UTP in the alpha position (pink is the radiolabeled)
Why not just radiolabel the reagent