Sigma54 is important in transcription initiation in cells. Sigma54/RNA polymerase holoenzyme forms a stable complex with promoter DNA which is incapable of initiation. The activator protein binds upstream of the promoter and contacts the closed promoter complex by DNA looping. The activator uses the energy from ATP hydrolysis to re-model the closed promoter complex, initiating transcription.
4 functional domains of sigma54: 1. binding to the activator protein 2. core RNAP binding domain 3. and 4. are the DNA binding domains Sigma54 has to initiate transcription down at the right end after binding/ATP hydrolysis of the activator at the left end.
We think that the mechanism is the second option, that a conformational change in sigma54 initiates transcription and that this conformational change is brought about after an activator protein binds and induces “tugging” on the molecule. The hinge point is evidence for this hypothesis.
In order to have a controlled, measurable experiment, you must attach your protein on both ends to a long (500bp) DNA via a disulfide bond. The DNA then must tightly interact with the polystyrene beads. A laser can be used to stretch the protein and the force can be measured.
Forces applied on the molecule are measured by momentum changes in the laser light beam
Honors thesis overview: Katie Amberg-Johnson
Use of Molecular Tweezers to Investigate Fracture Point of σ 54 Core Binding Domain Katie Amberg-Johnson College of Natural Resources University of California, Berkeley January, 2012
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