How does σ 54 initiate transcription on one end of the protein in response an activation on the other end of the protein? 1. In the absence of activator, activator binding domain inhibits open complex formation. A single ATP hydrolysis event is sufficient to allow open complex formation. 2. Multiple ATP hydrolysis cycles pull activator binding domain through the pore. This force causes a conformational change in the DNA binding domain that allows open complex formation.
Core Binding Domain is comprised of two subdomains Eunmi Hong, 2009 3 Helical Bundle 4 Helical Bundle
Step 1. Generate DNA handles Step 2. Express and purify CBD of σ 54 Step 3. Attach DNA handles to CBD -DTDP activation of CBD -Removal of DTT from DNA handles Step 4. Attach protein-DNA chimeras to polystyrene beads Step 5. Tug. My project is to simulate this proposed “tugging” by the Activator Binding Domain on the Core RNAP Binding Domain with the use of molecular tweezers
DNA Handles Primers : 5' thiol-GCT-ACC-GTA-ATT-GAG-ACC-AC with either 5' biotin-CAA-AAA-ACCCCT-CAA-GAC-CC or 5' digoxigenin-CAAAAA-ACC-CCT-CAA-GAC-CC Handles generated with standard PCR protocol except for the addition of 1M DTT and ending in a total of around 400 μg of each handle in 10mL. DNA purification was achieved using HI-Speed Plasmid-Midi Kit.
Step 1. Protein is denatured using guanidine hydrochloride and buffer exchanged into a buffer containing DTDP Step 2. DNA handles are buffer exchanged into a buffer containing no DTT using gravity filtration Step 3. DTDP bound protein is allowed to reacted with DNA handles over night Step 4. Protein is purified from unreacted DNA Attachment of DNA Handles to CBD
Molecular Tweezers Possibility A Possibility B - one rip, indicating CBD unfolds concertedly - two rips, indicating that the two subdomains unfold separately - supports hypothesis that physical stress causes conformational changes throughout the molecule to initiate transcription Force (pN) Force (pN) Extension Extension
Acknowledgments Professor Dave Wemmer Alex Siegel Dr. Bharat Jagannathan Dr. Christian Wilson Professor Wenshu Wang All members of the Wemmer lab Acknowledgments