Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 839-848 |
| Number of pages | 10 |
| Journal | Nucleic acids research |
| Volume | 36 |
| Issue number | 3 |
| DOIs | |
| State | Published - Feb 2008 |
Bibliographical note
Funding Information:We would like to thank Dr Wei Zhao and Nick Berge for technical assistance and Dr Jeffrey Lary for performing ultracentrifugation of RNAs and data analysis. Ultracentrifugation was performed at the University of Connecticut Analytical Ultracentrifugation Facility in Storrs, CT (James L. Cole, Director). Funding support has been given by National Institutes of Health (GM-059604 to S.G.; P41RR02301, P41GM66326 RR02781 and RR08438 to the National Magnetic Resonance Facility at Madison); Minnesota Medical Foundation (3484-9221-05 to H.M.); National Science Foundation (BIR-961477 to the NMR facility at University of Minnesota, DMB-8415048, OIA-9977486 and BIR-9214394 to the National Magnetic Resonance Facility at Madison). Funding to pay the Open Access publication charges for this article was provided by NIH grant number GM-059604.