Question: 2. Modified from Fickert and Mulller-Hill (J. Mol. Biol. 1992) Tetrameric Lac repressor binds to …

2. Modified from Fickert and Mulller-Hill (J. Mol. Biol. 1992)
Tetrameric Lac repressor binds to two sites, O1 and O2, in the Lac
operator to maximally repress transcription. Binding to both O1 and
O2 results in formation of a DNA loop that enhances repression.
[Hint: that means the tetramer can bind both Lac O1 and O2
simultaneously. Also, Lac O1 has a higher binding affinity for
repressor (KD = 1000 nM) than Lac O2 (KD = 100 nM).] Purified
tetrameric repressor was subjected to DNA mobility shift assays to
display interactions between Lac repressor and operator O1 and O2
(Figure 2, attached pdf). The mobility of free 32P-labeled 84-nt
Lac O1 DNA without added proteins is shown in lane 1. A
non-saturating amount of labeled Lac O1 was incubated with Lac
repressor in lane 3. In lane 5, an unlabeled Lac O2 fragment 24
nucleotides in length was added in vast molar excess compared to
22P-84 nt Lac O1 (Chase lac O DNA) that was already bound to Lac
repressor. The resulting protein/DNA complexes were separated by
polyacrylamide gel electrophoresis and auto-radiographed to
visualize the labeled DNA fragments. The gel slot shows the
positions where the samples were loaded; electrophoresis occurs
from top (anode) to bottom (cathode).

– What is the importance of showing lane 1 and fragment A?

– What does complex B represent in lanes 3 and 5? Diagram what
you think binding complex B looks like.

– What does complex C represent in lane 5? Include a diagram for
what you think binding complex C looks like.

– What is a reason for adding excess 24-nt Lac O2 fragment?