Models for Packaging of DNA by

It is generally understood that pRNA is part of an ATPase and that it possesses at least two conformations: relaxed and contracted. Driven by ATP hydrolysis, which causes an alternation between relaxed and contracted states, the individual parts of the hexameric pRNA complex help rotate the translocation machinery. gp16, a protein that binds ATP, is also involved in ATP hydrolysis.

The requirement of an intermolecular loop/loop interaction between individual pRNA molecules during DNA packaging has led to the belief that pRNA forms a hexamer (175,176) and supports a model incorporating the sequential action of pRNA. Individual pRNAs likely need to communicate with each other during DNA packaging to ensure that motion is consecutive. Inter-pRNA

Fig. 3. ^29 pRNA as polyvalent vehicles for delivery genes in therapy. pRNA multimers could be used to target a cell receptor and to co-deliver therapeutic siRNA, ribozymes or other biologically active molecules. Adapted from (Shu, Huang et al., 2003) with permission of J. Biol. Chemistry.

interactions via loops might serve as a link to pass signals to adjacent pRNAs, regulating sequential conformational changes and/or interactions. Thus, base pairing between the right- and left-hand loops might be necessary to transfer a conformational change from one pRNA to an adjacent one.

Several models for DNA translocation into the ^29 procapsid have been proposed (137,143,200-202). It is generally believed that DNA is translocated through the axial hole of the portal vertex, much like a threaded rod moving along a nut. Another model hypothesizes that supercoiled DNA wraps around the portal vertex and that its rotation allows DNA to pass into the procapsid via the outside of the vertex (202). In a model proposed in 1997 (143), sequential action of multiple pRNAs, in conjunction with other components, is vital to DNA packaging. Six copies of pRNA form a hexamer that interacts with the capsid pentamer and moves in discrete 12° steps (Fig. 3). Thirty ATPs are needed for each completed cycle of motor rotation. This model is in good agreement with existing 3D structural data obtained recently by crystallography (137), complementary modification, photoaffinity crosslinking, chemical modification, chemical modification interference, nuclease probing, AFM, and computer-modeling (158) methods.

pRNA sequence and secondly structure D Chimeric pRNA

Right hartf loop *

pRNA sequence and secondly structure D Chimeric pRNA

Right hartf loop *

Fig. 4. Sketch showing chimeric pRNA harboring a ribozyme. (A), The sequence and secondary structure of pRNA. The curved line points to the two interacting loops. (B), Structural outline of the pRNA/ribozyme chimera. A ribozyme targeting at the Hepatitis B virus mRNA is connected to both the 5' and 3'-end of pRNA. Adapted from (Hoeprich, Zhou et al., 2003) with permission of Nature Publisher.

Fig. 4. Sketch showing chimeric pRNA harboring a ribozyme. (A), The sequence and secondary structure of pRNA. The curved line points to the two interacting loops. (B), Structural outline of the pRNA/ribozyme chimera. A ribozyme targeting at the Hepatitis B virus mRNA is connected to both the 5' and 3'-end of pRNA. Adapted from (Hoeprich, Zhou et al., 2003) with permission of Nature Publisher.

In the model proposed in ref. 137, DNA located in the central channel interacts with one subunit of the portal. A 12° rotation of the narrow end of the connector leads to lengthwise expansion of the connector via a slight change in the angle of the long helices, and the wide end of the connector follows the narrow end. Such a "following" allows the structure to relax and contract while translating 2 bp of DNA into the capsid (137). In addition, the "motor-ratchet" model of DNA packaging has been proposed and reviewed recently (17). In this model, the connector rectifies DNA motion via either thermal, biased thermal, or oscillating processes. The motor-ratchet hypothesis has been widely used to interpret data pertaining to biochemistry, genetics, energetics, structure, and dynamics in packaging. The structure of both the connector and pRNA (158) exhibits orientational tilting toward one direction.

At least four distinct models can be proposed to describe the specific mechanisms underlying the translocation of DNA resulting from such motors, which are discussed next.

The force of DNA movement is Brownian motion (17,203,204). The motor is a ratchet (17) used simply to ensure that DNA moves in one direction, but not the opposite.

DNA is translocated through the axial hole of the portal vertex, much like a threaded rod processing through a nut, and thus DNA packaging is achieved by utilizing the "threaded" helical nature of dsDNA (142). The motor, composed of the pRNA and connector, operates like a hex nut to drive a bolt (143). Evidence for this model would include establishing that DNA translocates regularly at a fixed ratio of distance moved to the number of motor turns.

The fivefold/sixfold symmetry mismatch and sequential contraction and relaxation of motor components generates the force required for motor rotation (142,143). If this model is correct, discrete steps in 12° intervals in component rotation will be found.

DNA located in the central channel interacts with one subunit of the portal (137). A 12° rotation of the narrow end of the connector leads to lengthwise expansion of the connector via a slight change in the angle of the long helices; the wide end of the connector follows the narrow end. Such a "following" allows the structure to relax and contract while translating 2 bp of DNA into the capsid (137). This model implies that one complete turn of the connector transfers 60 bp of DNA into the procapsid.

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