Abstract
Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage r29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33-35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 9953-9963 |
| Number of pages | 11 |
| Journal | Nucleic acids research |
| Volume | 40 |
| Issue number | 19 |
| DOIs | |
| State | Published - Oct 2012 |
Bibliographical note
Funding Information:National Institutes of Health (NIH) [GM-059604 and GM-095516]. Funding for NMR instrumentation was provided by the Office of the Vice President for Research, the Medical School, the College of Biological Science, NIH, NSF and the Minnesota Medical Foundation; NIH [P41RR02301, P41GM66326, RR02781 and RR08438 to the National Magnetic Resonance Facility at Madison]; National Science Foundation [DMB-8415048, OIA-9977486 and BIR-9214394 to the National Magnetic Resonance Facility at Madison]. Funding for open access charge: NIH [GM-059604].