Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation.
Bacteriophage phi29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during maturation. An intriguing feature of phi29 assembly is that a virus-encoded RNA (pRNA) is required for the packaging of its genomic DNA. Psoralen cross-linking, primer extension, and T1 RNase partial digestion revealed that pRNA had at least two conformations; one was able to bind procapsids, and the other was not. In the presence of Mg2+, one stretch of pRNA, consisting of bases 31 to 35, was confirmed to be proximal to base 69, as revealed by its efficient cross-linking by psoralen. Two cross-linking sites in the helical region were identified. Mg2+ induced a conformational change of pRNA that exposes the portal protein binding site by promoting the refolding of two strands of the procapsid binding region, resulting in the formation of pRNA-procapsid complexes. The procapsid binding region in this binding-competent conformation could not be cross-linked with psoralen. When the two strands of the procapsid binding region were fastened by cross-linking, pRNA could neither bind procapsids nor package phi29 DNA. A pRNA conformational change was also discernible by comparison of migration rates in native EDTA and Mg2+ polyacrylamide gel electrophoresis and was revealed by T1 RNase probing. The Mg2+ concentration required for the detection of a change in pRNA cross-linking patterns was 1 mM, which was the same as that required for pRNA-procapsid complex formation and DNA packaging and was also close to that in normal host cells.
Chen
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Department of Pathobiology, Purdue University, West Lafayette, Indiana 47907, USA.
Guo
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D017103:Bacillus Phages / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D002213:Capsid / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004279:DNA, Viral / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005363:Ficusin / Q000737:chemistry; D008274:Magnesium / Q000494:pharmacology*; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D011498:Protein Precursors / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000187:drug effects*; D019065:Virus Assembly
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D011498:Protein Precursors; D012367:RNA, Viral; D008274:Magnesium; D005363:Ficusin