Question: Tetrameric Lac repressor binds to two sites, O1 and
O2, in the Lac operator to maximally repress
…
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?
