Abstract
Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA–gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a ‘Z’-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.
Highlights
Bacillus subtilis phage phi29 assembles the genome into a preformed protein capsid to near-crystalline density by a highly efficient molecular motor [1–3]
The phi29 prohead RNA (pRNA) domain II-tRNA crystal structure was solved at 3.3 Aresolution (Figure 1C and Supplementary Table S1) using molecular replacement and multiple rounds of fitting
We investigated the pRNA–gp16 complex structure through crystallography, small angle X-ray scattering (SAXS) and chemical probing
Summary
Bacillus subtilis phage phi assembles the genome into a preformed protein capsid (prohead) to near-crystalline density by a highly efficient molecular motor [1–3]. The phi motor core contains a ribonucleoprotein (RNP) complex of prohead RNA (pRNA) and gene product 16 (gp16) ATPase (Figure 1A) [5]. This RNP complex actively transports DNA inside the prohead capsid using energy derived from ATP binding and hydrolysis [6–8]. Located near the gp 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]. The molecular mechanism of this highly conserved domain II remained poorly understood as, far, it does not appear to participate in vitro DNA packaging or force generation
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