Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29

Abstract

During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864-3871] suggested that the foothold for pRNA was the connector and that the pRNA-connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS-PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA-connector complex and that the foothold for pRNA is the connector but not the capsid protein.

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

“…The ATPase must assemble to the head; in the other phages, the large subunit of the terminase interacts directly with the connector and perhaps the head shell, whereas in 29 it is the prohead binding domain of pRNA (Fig. 6C, blue shading) that binds directly to the head, interacting with the shell protein (14) and the connector (17)(18)(19). Prohead binding thereby positions the A-helices of pRNA (Fig.…”

“…1C) (16). It is a component of the prohead, making critical interactions with both the connector (17,18) and the head shell (14,19,20). Early showing the dodecameric connector (portal) in green, the pentameric pRNA ring in magenta, and the pentameric ring ATPase gp16 in blue.…”

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

“…The interaction between pRNA and the connector was demonstrated by crosslinking of the two (Garver and Guo 1997). It was found that the N-terminal 14 amino acids of gp10 are essential for pRNA binding since deletion of these 14 amino acids rendered the connector incompetent for RNA binding (Xiao et al 2005) and mutation in R3K4R5 led to a similar result (Atz et al 2007). Particularly, these three positively charged amino acids (R3K4R5) are believed to be responsible for the binding to pRNA with the negatively charged phosphate backbone.…”

Interaction of gp16 with pRNA and DNA for Genome Packaging by the Motor of Bacterial Virus phi29

“…17,41 A significant novelty of the phi29 DNA-packaging motor is that the motor involves an RNA molecule, 42 named pRNA, that forms a hexameric ring [43][44][45][46] and is attached to the N terminus of the connector protein gp10. 47 Originally, it was proposed that gp3 and gp16 were non-structural DNApackaging proteins. gp3, with its smaller molecular mass, was believed to be responsible for DNA binding, and the larger subunit, gp16, which contained the A-type and B-type sequences for ATP-binding, was believed to be responsible for procapsid binding.…”