Article

Modular assembly of chimeric phi29 packaging RNAs that support DNA packaging

Department of Chemistry, University of Southern California, LJS-251, 840 Downey Way, Los Angeles, CA 90089-0744, USA.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 09/2008; 372(4):589-94. DOI: 10.1016/j.bbrc.2008.05.094
Source: PubMed

ABSTRACT The bacteriophage phi29 DNA packaging motor is a protein/RNA complex that can produce strong force to condense the linear-double-stranded DNA genome into a pre-formed protein capsid. The RNA component, called the packaging RNA (pRNA), utilizes magnesium-dependent inter-molecular base-pairing interactions to form ring-shaped complexes. The pRNA is a class of non-coding RNA, interacting with phi29 motor proteins to enable DNA packaging. Here, we report a two-piece chimeric pRNA construct that is fully competent in interacting with partner pRNA to form ring-shaped complexes, in packaging DNA via the motor, and in assembling infectious phi29 virions in vitro. This is the first example of a fully functional pRNA assembled using two non-covalently interacting fragments. The results support the notion of modular pRNA architecture in the phi29 packaging motor.

Download full-text

Full-text

Available from: Peter Z Qin, Jul 14, 2015
0 Followers
 · 
92 Views
  • Source
    • "Previous results from various studies, including base deletion and mutation (Bailey et al. 1990; Reid et al. 1994c; Zhang et al. 1994, 1995b, 1997), ribonuclease probing (Reid et al. 1994a; Chen and Guo 1997), oligo targeting (Zhang et al. 1995a), competition assays to inhibit phage assembly (Trottier et al. 1996), UV crosslinking to portal protein (Garver and Guo 1997), psoralen crosslinking, and primer extension (Chen and Guo 1997), indicated two functional domains that could fold independently in pRNA. The two interlocking loops, along with the double-helical packaging domain for the binding of the motor ATPase (Lee et al. 2006), are connected through the 3WJ region in pRNA and a " three-arms around a hinge " model was proposed for pRNA function (Fang et al. 2008). Flexibility at the U72U73U74 bulge region was found to be essential for pRNA activity in gearing the DNA packaging motor (Reid et al. 1994c; Zhang et al. 1997), indicating that a conformational change may be required during DNA packaging. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg(2+). The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.
    RNA 07/2013; DOI:10.1261/rna.037077.112 · 4.62 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Many nucleic acid-binding proteins and the AAA+ family form hexameric rings, but the mechanism of hexamer assembly is unclear. It is generally believed that the specificity in protein/RNA interaction relies on molecular contact through a surface charge or 3D structure matching via conformational capture or induced fit. The pRNA of bacteriophage phi29 DNA-packaging motor also forms a ring, but whether the pRNA ring is a hexamer or a pentamer is under debate. Here, single molecule studies elucidated a mechanism suggesting the specificity and affinity in protein/RNA interaction relies on pRNA static ring formation. A combined pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group bind to the motor nonspecifically. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension or reduction of the ring circumference failed in motor binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and packaged DNA. The results confirmed the stoichiometry of pRNA on the motor was the common multiple of two and three, thus, a hexamer.
    Nucleic Acids Research 11/2008; 36(20):6620-32. DOI:10.1093/nar/gkn669 · 9.11 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nanobiotechnology involves the creation, characterization, and modification of organized nanomaterials to serve as building blocks for constructing nanoscale devices in technology and medicine. Living systems contain a wide variety of nanomachines and highly ordered structures of macromolecules. The novelty and ingenious design of the bacterial virus phi29 DNA packaging motor and its parts inspired the synthesis of this motor and its components as biomimetics. This 30-nm nanomotor uses six copies of an ATP-binding pRNA to gear the motor. The structural versatility of pRNA has been utilized to construct dimers, trimers, hexamers, and patterned superstructures via the interaction of two interlocking loops. The approach, based on bottom-up assembly, has also been applied to nanomachine fabrication, pathogen detection and the delivery of drugs, siRNA, ribozymes, and genes to specific cells in vitro and in vivo. Another essential component of the motor is the connector, which contains 12 copies of a protein gp10 to form a 3.6-nm central channel as a path for DNA. This article will review current studies of the structure and function of the phi29 DNA packaging motor, as well as the mechanism of motion, the principle of in vitro construction, and its potential nanotechnological and medical applications.
    Annals of Biomedical Engineering 10/2009; 37(10):2064-2081. DOI:10.1007/s10439-009-9723-0 · 3.23 Impact Factor
Show more