Journal
NUCLEIC ACIDS RESEARCH
Volume 40, Issue 19, Pages 9953-9963Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gks724
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Funding
- National Institutes of Health (NIH) [GM-059604, GM-095516]
- Office of the Vice President for Research, the Medical School, the College of Biological Science, NIH, NSF
- Minnesota Medical Foundation
- NIH [GM-059604, P41RR02301, P41GM66326, RR02781, RR08438]
- National Science Foundation [DMB-8415048, OIA-9977486, BIR-9214394]
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Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage phi 29 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 degrees 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.
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