Mechanism and consequences of Mu transpososome remodeling by the ClpX chaperone

E. coli ClpX is a member of the Clp/Hsp100 family of ATPases that remodel multi-component complexes and facilitate ATP-dependent protein degradation. Protein remodelers alter the biological activity of their substrates, typically by changing the quaternary structure of their target proteins. ClpX remodels protein-DNA complexes, termed transpososomes, made during recombination of the phage Mu. When recombination is complete, the core four-subunit transpososome complex does not spontaneously release the DNA; transposase remains so stably bound that subsequent replication of the Mu genome is inhibited. To understand how ClpX releases the replication block without destroying the transpososomes, we characterized the mechanism and products of transpososome remodeling. To better understand the mechanism ClpX uses to facilitate remodeling, I first participated in a collaborative project that defined major biochemical reaction steps involved in protein degradation by ClpX and its associated peptidase ClpP. These experiments demonstrated that ClpX acts as a powerful unfolding enzyme during degradation reactions. Then, I asked whether this same unfolding activity was also responsible for transpososome remodeling. These experiments demonstrated that ClpX can unfold transposase monomers, and that it exerts a conformational change in transposase subunits during remodeling. However, only subunits directly contacted by ClpX undergo a conformational change. Analysis of the minimal interaction necessary between ClpX and the complex revealed that recognition of one subunit in the complex is sufficient for remodeling.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2003

Author dc:creator

• Burton, Briana M. (Briana Marie), 1977-

Advisor dc:contributor.advisor

• Tania A. Baker.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/29290

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/29290