Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Illuminating Obscure States of the Phage T3 DNA Packaging Motor
We use DNA packaging of the related phages, T3 and T7, as a model to develop analysis of biological structure-function relationships. A key objective is characterization of the DNA packaging motor in its various active states. We advance by improving isolation of DNA packaging intermediates generated in vivo and by improving structural characterization of isolated intermediates. A pre-assembled procapsid, called capsid I for T3/T7, initiates packaging (Guo et al., 2013). Capsid I has a packaging ATPase-nuclease (terminase) attached to a 12-fold connector that is embedded in the shell. Early in packaging, capsid I converts to a more angular, larger, mature phage-like capsid called capsid II. Most investigators assume that the capsid II-associated, DNA-containing shell is non-dynamic during DNA packaging; the same is assumed for analogous capsids of other phages. However, we have found that the icosahedral shell of capsid II has an average radius that decreases by 1.4 %, via rigid body rotation of the asymmetric subunit, when it converts to the mature phage capsid (Guo et al., 2014). The mature capsid of a tail-free phage T3 head (30 nm in radius) contracts when it partially expels DNA in quantized lengths (Serwer et al., 2014). In the current study, we use (1) genetics via directed evolution and (2) hydration-based buoyant density centrifugation to isolate a previously obscure T3 intermediate that has incompletely packaged DNA and a capsid with a Nycodenz- and negative stain (PTA)-impermeable shell that varies in appearance. This intermediate is, therefore, thought to be in a structurally dynamic state. The shell radius varies from hyper-expanded to contracted, and can be driven by ATP to favor a contracted state. A previous hypothesis (Serwer et al., 2011) has predicted these states via proposal that the motor has a back-up, expansion-contraction (type 2) cycle that activates when the terminase-driven (type 1) cycle becomes irreversibly stalled.
Supported by the Welch Foundation (AQ-764 to PS) and NIH (R01AI072035 and S10RR023011 to WJ).
F. Guo, Z. Liu, P.-A. Fang, Q. Zhang, E.T. Wright, W. Wu, C. Zhang, F. Vago, Y. Ren, J. Jakana, W. Chiu, P. Serwer & W. Jiang. (2014) Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions. Proc. Natl. Acad. Sci., USA 111, 6811-6816.
P. Serwer, E.T. Wright, Z. Liu Z & W. Jiang. (2014) Length quantization of DNA partially expelled from heads of a bacteriophage T3 mutant. Virology 456-457, 157-170.
P. Serwer. (2011) Proposed ancestors of phage nucleic acid packaging motors (and cells). Viruses 3, 1249-1280.
1Department of Biochemistry