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CPLC Seminar: "Shining a light: Using single-molecule fluorescence to decipher the coordination between subunits of the bacteriophage T4 DNA packaging motor"

SpeakerVishal Kottadiel, Department of Biology (Rao Lab), Catholic University of America, Washington, DC
Date Nov 2, 2012
Time 2:30 pm - 3:00 pm  
Location 144 Loomis
Sponsor Center for the Physics of Living Cells
Contact Shawn McCormick
Event type CPLC Seminar
Views 1160

During viral infection, the tailed dsDNA bacteriophage T4 employs a powerful, ATP-dependent molecular motor to translocate its 171-kb genome into a preformed icosahedral head. The motor is a homopentamer comprising five copies of the large terminase protein, gp17, and attaches to the head via a special vertex formed by the dodecameric portal protein, gp20. Previous single molecule studies have shown that the motor can package DNA at speeds up to 2000 bp/s, generating forces in excess of 60 pN, making it one of the fastest and most powerful packaging motors reported to date. An open question on the mechanism of T4 packaging is how the five gp17 subunits are coordinated during translocation, or whether all subunits of the pentamer are even necessary to translocate DNA. Bulk mutant poisoning studies suggest the packaging motor can tolerate up to one non-functional subunit in the pentameric ring and still package DNA. The mechanism by how this presumably occurs is not understood. In this study, we aim to use optical tweezers simultaneous with single-molecule fluorescence as well as TIRF microscopy in order to shed light on the coordination of the T4 packaging motor. Our plan is to assemble gp17 pentamers containing a mix of wildtype and “dead”, fluorescently labeled mutant subunits and to monitor their packaging activity at the single-molecule level. The fluorescence will enable us to determine directly the stoichiometry of dead mutant vs. wildtype subunits in each pentamer and correlate it to packaging activity. Implications of these studies to the current model of the packaging mechanism will be discussed.

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