Experimental Attempts to Elucidate Motor Mechanism of

3.1.1. Establishment of a Highly Sensitive In Vitro Packaging System

Utilizing pRNA's readily manipulable structure, a defined in vitro §29 DNA-packaging system has been developed (9). With purified procapsid, gp16, pRNA, and ATP, up to 90% of the added DNA-gp3 can be packaged into the procapsid via the motor constructed with recombinant gene products. After DNA packaging, the in vitro assembly system can convert a DNA-filled capsid into infectious §29 virions with the addition of purified proteins gp9, gp11, gp12, and gp13 (103,104). With this system, up to 5 x 109 infectious virions/mL can be obtained in the presence of all required components, yet not a single infectious virion is detected when even one essential component is absent. This system, with a sensitivity of eight orders of magnitude, has been used to assay the function of the DNA-packaging motor (180,192).

3.1.2. Circularly Permuted pRNA

An important approach in analyzing pRNA function has been the construction of circularly permuted pRNAs (cp-pRNAs), in which any internal base of the pRNA could be reassigned to serve as new 5'- or 3'-termini (169,193). Two tandem pRNA coding sequences separated by a 3- or 17-base loop sequence were cloned into a plasmid. Polymerase chain reaction (PCR) primer pairs such as P6/P5, complementary to various locations within the tandem pRNA coding sequences, were designed to synthesize PCR fragments for the transcription of cp-pRNAs. Results indicated that neither the small nor the large loop interferes with biological activity of the molecule. It has been demonstrated that nonessential bases or ones adjacent to them can be used as new termini for constructing active cp-pRNA; this system greatly facilitates the construction of mutant pRNA via PCR and enables the labeling of any specific internal base by radioisotopes, fluorescence (194), or photoaffinity agents.

3.1.3. Computer Modeling

The goal of modeling pRNA structure is to organize collections of structural data from crosslinking, chemical or ribonuclease probing, chemical modifica-

Fig. 2. Computer models of pRNA hexamer and the motor complex of bacterial virus ^29. (A), Hexamer shows the connector binding domain in green, as well as the DNA translocating domain colored red (5'-end) and cyan (3'-end). The DNA translocating domain of the 5'/3'-paired region points up. (B), The crystal structure of the connector (From refs. Valle, Kremer et al., 1999; Valpuesta, Fernandez et al., 1999). The RNA Recognition Motif is blue. (C), Docking of the pRNA hexamer to the RNA binding domain of the connector. (D), Procapsid Bottom view. Adapted from (Shu, Huang et al., 2003) with permission of J. Biol. Chemistry.

Fig. 2. Computer models of pRNA hexamer and the motor complex of bacterial virus ^29. (A), Hexamer shows the connector binding domain in green, as well as the DNA translocating domain colored red (5'-end) and cyan (3'-end). The DNA translocating domain of the 5'/3'-paired region points up. (B), The crystal structure of the connector (From refs. Valle, Kremer et al., 1999; Valpuesta, Fernandez et al., 1999). The RNA Recognition Motif is blue. (C), Docking of the pRNA hexamer to the RNA binding domain of the connector. (D), Procapsid Bottom view. Adapted from (Shu, Huang et al., 2003) with permission of J. Biol. Chemistry.

tion interference, cryo-AFM, and other genetic data into a 3D form. Because a large number of structural constraints are available, computer programs can successfully construct 3D structures (158,195,196) (Fig. 2).

X-ray crystallography studies have revealed that the ^29 connector contains three sections: a narrow end, a central section, and a wider end, with diameters of 6.6, 9.4, and 13.8 nm, respectively (136,137,197). The hexameric pRNA model by Hoeprich and Guo (158) describes a central channel with a diameter of 7.6 nm that perhaps can sheath onto the narrow end of the connector to be anchored by the central section of the connector, which is wider than the central channel of the pRNA hexamer.

The connector-binding domain is located in the middle of the pRNA primary sequence (i.e., bases 23-97), and the DNA-translocation domain is located at the 5'/3' paired ends. It has been predicted that the connector protein (gp10) contains a conserved RNA recognition motif (RRM), located between residues 148 to 214 of each gp10 monomer. This region of gp10 is located at the narrow end of the dodecameric connector that protrudes from the procapsid (156,198). The hexamer model by Hoeprich and Guo (158) agrees with the aforementioned data, showing that pRNA bases 23 to 97 within the connector-binding domain interact with the predicted RRM motifs of the connector, and the 5'/3' paired region, comprising the DNA-translocation domain, extends away from the connector.

3.1.4. Use of Tweezers to Determine Force of Motor

Optical tweezers offer an interesting method by which to measure packaging force and speed (199). In this procedure, partly packaged motor complex has a microsphere attached to it via unpackaged DNA. This microsphere is caught in an optical trap and tethered to a second bead. When ATP is introduced and packaging initiates, the two beads move closer together. The amount of DNA tension is monitored, and from this, the bead displacement can be calculated; various measurements of packaging dynamics are then possible, including an examination of the presence of packaging "slips" and "pauses," where irregularities occur in packaging speed. With additional load, the force needed to prevent the DNA from being inserted is about 57 pN, indicating that the §29 DNA-packaging motor is the strongest biomotor studied to date. Using this method, it has been possible to determine the speed of §29 DNA packaging, which is initially about 100 bases/s, gradually slowing to a halt as the capsid is filled (199). The fivefold/sixfold symmetry mismatch between the procapsid and pRNA has been cited to help explain the mechanism of motor rotation (142,143,180), but the precise mechanism by which DNA moves into the capsid during packaging has yet to be elucidated.

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