title: Mapping the inter-RNA interaction of bacterial virus phi29 packaging RNA by site-specific photoaffinity cross-linking.
abstract: During replication, the lengthy genome of double-stranded DNA viruses is translocated with remarkable velocity into a limited space within the procapsid. The question of how this fascinating task is accomplished has long been a puzzle. Our recent investigation suggests that phi29 DNA packaging is accomplished by a mechanism similar to the driving of a bolt with a hex nut and that six packaging RNAs (pRNAs) form a hexagonal complex to gear the DNA-translocating machine (Chen, C., and Guo, P. (1997) J. Virol. 71, 3864-3871; Zhang, F., Lemieux, S., Wu, X., St.-Arnaud, S., McMurray, C. T., Major, F., and Anderson, D. (1998) Mol. Cell 2, 141-147; Guo, P., Zhang, C., Chen, C., Garver, K., and Trottier, M., (1998) Mol. Cell 2, 149-155). In the current study, circularly permuted pRNAs were used to position an azidophenacyl photoreactive cross-linking agent specifically at a strategic site that was predicted to be involved in pRNA-pRNA interaction. Cross-linked pRNA dimers were isolated, and the sites of cross-link were mapped by primer extension. The cross-linked pRNA dimer retained full activity in phi29 procapsid binding and genomic DNA translocation, indicating that the cross-link distance constraints identified in dimer formation reflect the native pRNA complex. Both cross-linked dimers either containing or not containing the interlocking loops for programmed hexamer formation bound procapsid equally well; however, only the one containing the interlocking loops programmed for hexamer formation was active in phi29 DNA packaging. The cross-linked pRNA dimers were also identified as the minimum binding unit necessary for procapsid binding. Primer extension of the purified cross-linked pRNA dimers revealed that base G(82) was cross-linked to bases G(39), G(40), A(41), C(49), G(62), C(63), and C(64), which contribute to the formation of the three-way junction, suggesting that these bases are proximate in the formation of pRNA tertiary structure. Interestingly, the photoaffinity agent in the left interacting loop did not cross-link directly to the right loop as expected but cross-linked to bases adjacent to the right loop. These data provide a background for future modeling of pRNA tertiary structure.
authors.affiliation: Department of Pathobiology, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: K
authors.initials: K
authors.lastname: Garver
authors.forename: P
authors.initials: P
authors.lastname: Guo
mesh_terms: D017103:Bacillus Phages / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019920:Photoaffinity Labels / Q000737:chemistry*; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019065:Virus Assembly / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D019920:Photoaffinity Labels; D012367:RNA, Viral