Use of Molecular Tweezers to Investigate Fracture

Point of σ54 Core Binding Domain

Katie Amberg-JohnsonCollege of Natural Resources

University of California, BerkeleyJanuary, 2012

Transcription Initiation by σ54

Natasha Vidangos, 2010

How does σ54 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 σ54

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 400μg of each handle in 10mL.

DNA purification was achieved using HI-Speed Plasmid-Midi Kit.

Protein Preparation

Attachment of DNA handles to CBD

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 WemmerProfessor Dave WemmerAlex SiegelAlex SiegelDr. Bharat JagannathanDr. Bharat JagannathanDr. Christian WilsonDr. Christian WilsonProfessor Wenshu WangProfessor Wenshu WangAll members of the Wemmer labAll members of the Wemmer lab

Acknowledgments