Characterization of the Characterization of the Bacteriophage Bacteriophage ΦΦ29 Nanomotor29 NanomotorMay 1, 2009Pearl C. Pfiester

OutlineOutline Purpose Discovery of pRNA Overview The structural nature of pRNA Proposed models of pRNA activity Nanomotor Innovation

PurposePurpose

To present the evolution of understanding the Bacillus subtilis bacteriophage phi29 viral nanomotor structure◦ Phi29 selected by NIH as the model◦ Prominent scientist Peixuan Guo

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

•RNases inhibited phi29 assembly

•RNA-DNA hybridization showed that RNA component is viral and located in Hae II-H fragment

OverviewOverview

pRNA

?gp 16

gp 3

Number of CopiesNumber of Copies “Single-molecule dual

viewing total internal reflection fluorescence imaging system” or SMDV-TIRF◦ Can directly count the

number of copies of labeled pRNA by photobleaching fluorophores

Number of CopiesNumber of Copies Counting pRNA molecules

before initiation and during DNA translocation was counted

Before initiation: revealed a 6 steps of photobleaching◦ Procapsid/pRNA complexes

labeled Cy3 (green) During active translocation:◦ Procapsid/pRNA complexes

labeled Cy3 (green) and DNA labeled Cy5 (red)

◦ Only yellow spots were examined

◦ Revealed that pRNA is a hexamer during DNA translocation 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 CopiesNumber of Copies

Procapsid/pRNA complexes constructed with RNA dimers ◦ pRNA I and pRNA II was

labeled Cy3 (green) and Cy5 (red)

Most procapsid/pRNA complex spots were yellow

Yellow spots were plotted to show photobleaching steps for Cy3 and Cy5 separately

Each procapsid/pRNA complex had three Cy3 pRNA I and three Cy5 pRNA II molecules

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 ConfirmedNumber of Copies Confirmed Ferritin attached to

each pRNA at the 5’ end

Procapsids with six ferritin molecules were directly observed by electron microscopy

Figure 4. (Shu, Zhang, Jin, & Guo, 2007). Negatively stained EM images of pRNA conjugated with ferritin.

Structure of pRNAStructure of pRNA

How is the pRNA hexamer built?

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 Do pRNA pairs form dimers or form dimers or trimers?trimers?

Closed circle groups are more stable

Dimers are more stable than trimers

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.

Existence of dimers and trimers confirmed by cyro-atomic force microscopy (cryo-AFM)

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

Dimeric pRNAs constructed in different combinations◦ Covalently fused dimer

active in vitro◦ Base pairing between

right and left loops are critical for dimer and hexamer formation◦ Results suggest fused

tandem pRNAs are the building blocks of hexamers

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

SummarySummary pRNA is an integral component of DNA

translocation pRNA is a hexamer ring composed of 3 dimers

that stay together during active packaging Hexamer ring of pRNAs interacts with the

pentagonal opening of procapsid◦ Could mean motor rotation due to symmetry

mismatch◦ Which molecule is acting as rotor or stator?

Proposed ModelsProposed Models

Connector Rotating Thread Model◦ Rotation of gp16 is the driving

force for DNA translocation◦ DNA acts as threaded bolt

moving through the nut (rotating connector)◦ Requires perfect fit between

connector and DNA◦ No evidence for validation

See Movie:

http://bilbo.bio.purdue.edu/~viruswww/Rossmann_home/movies/phi29rot.php

Proposed ModelsProposed ModelsSequential action model Relative motion of the hexameric ring and pentagonal vertex of the

capsid could provide torque by ATP hydrolysis to rotate the motor as a whole◦ Like a six-cylinder car engine, sequential firing of six pRNAs could

rotate the motor by alternating contraction and relaxation conformational changes

This model is supported:◦ ATP induces conformational changes in pRNA◦ complete loss of packaging by pRNA mutations◦ replacing a subunit of pRNA with an inactive variant blocked packaging

motor function thereby supporting the speculation that each subunit in pRNA takes turns in packaging

Presently, there is no direct evidence against the model, but the direct observation of sequential versus random action of pRNAs has not been verified

Nanomotor InnovationNanomotor Innovation Understanding the packaging motor has:◦ Inspired the development of biomimetrics in

nanotechnology includes active or passive drug delivery or gene

therapy, tissue/organ repair, diagnosis of diseases by delivery of radioactive markers, or potential treatment for cancer and viral infection

◦ It can be used to construct nanomaterials ◦ Finally, this technology can create inexpensive and

highly sensitive analytical tools for probing and DNA recognition for sequencing by the electrical signals generated from the base pairs going through the pore

Literature CitedLiterature CitedChen 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.

SummarySummary

pRNA is an integral component of DNA translocation

pRNA is a hexamer ring composed of 3 dimers that stay together during active packaging

pRNA interacts with pentagonal opening of procapsid◦ Could mean motor rotation due to symmetry mismatch◦ Which molecule is acting as rotor or stator?