Mechanism and Dynamics of the Bacteriophage T4 DNA Packaging Motor

URI / Handle

Bacteriophage T4 is a tailed dsDNA virus that infects E.coli in order to replicate. A critical step in its life cycle is the translocation or "packaging" of its ~171Kb dsDNA genome into a preformed empty icosahedral head via an ATP-dependent process within the span of 3 to 5 minutes. It accomplishes this via a motor comprising the dodecameric portal protein gp20 and the pentameric large terminase protein gp17. The T4 motor is capable of translocating DNA at speeds up to 2000 bp/s whilst exerting forces in excess of 60 pN, making it the fastest and most powerful packaging motor studied to date. Although packaging in T4 has been well studied, the precise mechanism by which the motor functions is poorly understood, and is the focus of these studies.We show that the initiation pathway of packaging displays much plasticity with the motor even being able to package DNA into mature, once-filled heads. While bulk assays demonstrate that packaging efficiency decreases as ATP concentration is lowered, single molecule experiments show that the main cause for low packaging efficiency is due to an increase in number of pauses as ATP concentration is lowered rather than a reduction in translocation speed. Furthermore, DNA gets "unpackaged" during pause events, a slow, controlled release of DNA from the capsid, a feature that seems unique to the T4 system. Introduction of ATP analogs also causes pausing, although no unpackaging is associated with these pauses. Bulk experiments also show that the packaging motor can still translocate DNA despite the presence of a non-functional gp17 subunit within the pentameric ring. Single molecule experiments show that such motors exhibit frequent pausing during packaging. Similar to the pauses observed with ATP analogs, these pauses show no unpackaging.Our results point to a dynamic mechanism of packaging that allows the motor to overcome obstacles in initiation, cellular ATP concentrations, as well as motor composition in order to produce viable, infectious phage particles. We propose a four-state model for the T4 DNA packaging machine that involves tight co-ordination between DNA and the motor during packaging, and exhibits distinct packaging dynamics, random pausing and unpackaging, suggesting a novel mechanism for motor-DNA interaction and its coupling to ATP, different than that proposed for other packaging motors.
Details
Title

Mechanism and Dynamics of the Bacteriophage T4 DNA Packaging Motor

Contributors

Rao, Venigalla B (Advisor); Kottadiel, Vishal Isaac (Author); Golin, John E (Other); Tuma, Pamela L (Other); Greene, James J (Other)

Degree Information

Degree awarded: Ph.D. Biology. The Catholic University of America

Extent

133 p.

URI / Handle

http://hdl.handle.net/1961/10258

Advisor

Rao, Venigalla B

Other

Golin, John E; Tuma, Pamela L; Greene, James J

Subject

Virology; Biology; Biophysics; bacteriophage T4; molecular motor; optical tweezers; single molecule; viral DNA packaging

Accessioned

2012-06-01T16:44:11Z

Available

2012-06-01T16:44:11Z

Created

2012-01-01T00:00:00Z

Issued

2012-06-01T00:00:00Z

Abstract

Bacteriophage T4 is a tailed dsDNA virus that infects E.coli in order to replicate. A critical step in its life cycle is the translocation or "packaging" of its ~171Kb dsDNA genome into a preformed empty icosahedral head via an ATP-dependent process within the span of 3 to 5 minutes. It accomplishes this via a motor comprising the dodecameric portal protein gp20 and the pentameric large terminase protein gp17. The T4 motor is capable of translocating DNA at speeds up to 2000 bp/s whilst exerting forces in excess of 60 pN, making it the fastest and most powerful packaging motor studied to date. Although packaging in T4 has been well studied, the precise mechanism by which the motor functions is poorly understood, and is the focus of these studies.We show that the initiation pathway of packaging displays much plasticity with the motor even being able to package DNA into mature, once-filled heads. While bulk assays demonstrate that packaging efficiency decreases as ATP concentration is lowered, single molecule experiments show that the main cause for low packaging efficiency is due to an increase in number of pauses as ATP concentration is lowered rather than a reduction in translocation speed. Furthermore, DNA gets "unpackaged" during pause events, a slow, controlled release of DNA from the capsid, a feature that seems unique to the T4 system. Introduction of ATP analogs also causes pausing, although no unpackaging is associated with these pauses. Bulk experiments also show that the packaging motor can still translocate DNA despite the presence of a non-functional gp17 subunit within the pentameric ring. Single molecule experiments show that such motors exhibit frequent pausing during packaging. Similar to the pauses observed with ATP analogs, these pauses show no unpackaging.Our results point to a dynamic mechanism of packaging that allows the motor to overcome obstacles in initiation, cellular ATP concentrations, as well as motor composition in order to produce viable, infectious phage particles. We propose a four-state model for the T4 DNA packaging machine that involves tight co-ordination between DNA and the motor during packaging, and exhibits distinct packaging dynamics, random pausing and unpackaging, suggesting a novel mechanism for motor-DNA interaction and its coupling to ATP, different than that proposed for other packaging motors.

Type

Text; Dissertation

Local

Kottadiel_cua_0043A_10333

Publisher

The Catholic University of America

Publish Date

2012-06-01

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