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Nucleic Acids Research Advance Access published online on December 15, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm874
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Analysis of intermolecular base pair formation of prohead RNA of the phage ø29 DNA packaging motor using NMR spectroscopy

Aya Kitamura1, Paul J Jardine2, Dwight L Anderson2,3, Shelley Grimes2 and Hiroshi Matsuo1,*

1Department of Biochemistry, Molecular Biology and Biophysics, 2Department of Diagnostic and Biological Sciences and 3Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, USA

*To whom correspondence should be addressed. Tel: 612 626 6524; Fax: 612 625 2163; Email: matsu029{at}umn.edu

Received August 3, 2007. Revised September 25, 2007. Accepted September 30, 2007.

The bacteriophage ø29 DNA packaging motor that assembles on the precursor capsid (prohead) contains an essential 174-nt structural RNA (pRNA) that forms multimers. To determine the structural features of the CE- and D-loops believed to be involved in multimerization of pRNA, 35- and 19-nt RNA molecules containing the CE-loop or the D-loop, respectively, were produced and shown to form a heterodimer in a Mg2+-dependent manner, similar to that with full-length pRNA. It has been hypothesized that four intermolecular base pairs are formed between pRNA molecules. Our NMR study of the heterodimer, for the first time, proved directly the existence of two intermolecular Watson–Crick G–C base pairs. The two potential intermolecular A–U base pairs were not observed. In addition, flexibility of the D-loop was found to be important since a Watson–Crick base pair introduced at the base of the D-loop disrupted the formation of the intermolecular G–C hydrogen bonds, and therefore affected heterodimerization. Introduction of this mutation into the biologically active 120-nt pRNA (U80C mutant) resulted in no detectable dimerization at ambient temperature as shown by native gel and sedimentation velocity analyses. Interestingly, this pRNA bound to prohead and packaged DNA as well as the wild-type 120-nt pRNA.


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