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

Abstract

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage 29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of 29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left-and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although 29 differs from other doublestranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During viral assembly, double-stranded DNA (dsDNA) viruses, such as the tailed bacteriophages and herpesviruses, package their genomic DNA into a preformed viral shell (prohead) (1, 2). This process is driven by a viral genome-encoded molecular motor that assembles at a unique vertex of the prohead and utilizes the energy from ATP binding and hydrolysis to translocate DNA. During this process, the DNA is compacted to a nearly crystalline density and the motor must overcome the barriers of electrostatic repulsion and entropy that oppose this process. Indeed, the dsDNA viral packaging motors are among the most powerful molecular motors yet characterized (3).The packaging process involves distinct phases (Fig. 1A), including (i) initiation, which is comprised of assembly events that culminate in a prohead-motor-DNA substrate complex primed for packaging; (ii) translocation, whereby the DNA is driven into the head in an ATP-dependent manner; and (iii) termination, when DNA translocation ceases and the DNA-filled head is stabilized while the transient parts of the motor are readied for disassembly (2). While the mechanics of translocation are repeated over thousands of mechanochemical cycles, it is the singular events of the initiation phase that serve to provide the specificity for...

ATP/ADP modulates gp16–pRNA conformational change in the Phi29 DNA packaging motor

“…The remaining helices, P B and L BF , are recognized by ATPase gp16. This layout suggests that these conserved internal structures may interact with other viral components (such as gp3) as suggested previously (24,60).…”

“…Wild-type phi29 ATPase gp16 coding sequence was inserted into a pET-28 based SUMO recombinant expression vector. The SUMO-gp16 plasmid was transformed into Escherichia coli BL21(DE3) strain and shaken at 200 rpm in LB medium with kanamycin antibiotics at 37°C overnight (24). Then, the culture was diluted 1/100 in fresh LB medium.…”

“…SHAPE probing and data analysis were performed as previously described (17,41–44). The components for prohead-pRNA complex was prepared as described (24,45). The pRNA-free proheads incubated with wild-type 174-base pRNA in 25 mM Tris-HCl, pH 7.5–7.9, 5 mM MgCl 2 , 50 mM NaCl for 15 min at room temperature.…”

“…The resulting ring structure interfaces with the connector protein ring and head shell via a prohead binding subdomain (17,22,23), while the ATPase gp16 binds pRNA via the A-helix subdomain (16). Located near the gp16 ATPase-binding subdomain, the pRNA domain II imparts the specificity and stringency to the packaging process, restricting packaging to only one DNA fragment with fixed orientation in packaging polarity (18,24). However, the molecular mechanism of this highly conserved domain II remained poorly understood as, thus far, it does not appear to participate in vitro DNA packaging or force generation.…”

NMR Structures and Dynamics in a Prohead RNA Loop that Binds Metal Ions

“…1 Although the role of pRNA in the mechanism of the motor is poorly understood, mutations in the pRNA abolish packaging activity. 2 The A-helix of pRNA has a role in the initiation phase of packaging, 3 but the role of pRNA during packaging and the mechanism of the ATPase remain active areas of experiment and debate. 4–7 High-resolution structural data is necessary to determine the packaging motor mechanisms.…”