Characterization of the phi29 Bacteriophage Nanomotor

Characterization of the Bacteriophage Φ 29 Nanomotor May 1, 2009 Pearl C. Pfiester

Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Purpose ,[object Object],[object Object],[object Object]

Figure 1. (Guo, Erickson, & Anderson, 1987) (A) Restriction maps of the left end of phi29 for restriction enzymes. (B) Restriction fragment designations of (C). (D) Autoradiograph of a Southern blot of fragments in (C) after hybridization with [32P]RNA probe that was purified from prohead. Discovering pRNA ,[object Object],[object Object]

Number of Copies ,[object Object],[object Object]

Number of Copies ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Figure 2. (Shu, Zhang, Jin, & Guo, 2007). Experimental set up for single-molecule imaging. (A) pRNA labeled with fluorescent label. (B) Experimental design for before initiation of DNA translocation and during translocation (C).

Number of Copies ,[object Object],[object Object],[object Object],[object Object],[object Object],Figure 3. (Shu, Zhang, Jin, & Guo, 2007). Dual-view imaging of procapsid/pRNA complexes containing dimers labeled Cy3 pRNA I and Cy5 pRNA II. (A) Dimer with both labels. (B) Fluorescence image of procapsids dual-labeled dimers. (C) Comparison of empirical and predicted distribution of photobleaching steps of Cy3 pRNA I. (D) Florescence intensity versus time to show photobleaching steps which shows each procapsid/pRNA complex had three Cy3 pRNA I and three Cy5 pRNA II.

Number of Copies Confirmed ,[object Object],[object Object],Figure 4. (Shu, Zhang, Jin, & Guo, 2007). Negatively stained EM images of pRNA conjugated with ferritin.

Structure of pRNA ,[object Object],Figure 5. (Chen, Sheng, Shao, & Guo, 2000). (I) Wild type of pRNA. (II) One of the circularly permutated pRNA or cpRNA of the right and left hand loops. (III) Hexameric pRNA formed by hand-in-hand interaction between loops from pRNA pairs.

Do pRNA pairs form dimers or trimers? ,[object Object],[object Object],Figure 6. (Chen, Sheng, Shao, & Guo, 2000). (A) Polyacrylamide gel showing monomers (M), dimers (D) and trimers (T) in lanes 1-10. Lanes 9 and 10 showed isolation of dimers and trimers and then subjected to 10% denaturing gels in lanes 11-13. (B) Purified dimers and trimers were compared after gradient sedimentation.

[object Object],Figure 7. (Chen, Sheng, Shao, & Guo, 2000). Cyro-AFM images of pRNA dimers, monomers, and trimers.

[object Object],[object Object],[object Object],[object Object],Figure 8. (Chen, Sheng, Shao, & Guo, 2000). Packaging activities of pRNA dimers.

Summary ,[object Object],[object Object],[object Object],[object Object],[object Object]

Proposed Models ,[object Object],[object Object],[object Object],[object Object],[object Object],See Movie: http://bilbo.bio.purdue.edu/~viruswww/Rossmann_home/movies/phi29rot.php

Proposed Models ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Nanomotor Innovation ,[object Object],[object Object],[object Object],[object Object],[object Object]

Literature Cited Chen C., Sheng S., Shao Z., & Guo P. (2000). A dimer as a building block in assembling RNA. Journal of Biological Chemistry . 23: 17510-17516. Guo P., Erickson S., & Anderson D. (1987). A small viral RNA is required for in vitro packaging of bacteriophage φ29 DNA. 236(4802): 690-694. Guo P. & Lee T. (2007). Viral nanomotors for packaging of dsDNA and dsRNA. Molecular Microbiology . 64(4): 886-903. Review Article. Shu D., Zhang H., Jin J., & Guo P. (2007). Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system. EMBO Journal . 26: 527-537.

Characterization of the phi29 Bacteriophage Nanomotor

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