Macromolecular interactions of the bacteriophage phi29 pRNA required for DNA packaging
Abstract
Bacteriophage phi29 utilizes a viral-encoded RNA (pRNA) to insert its genomic DNA into the viral procapsid. Probing the pRNA with antisense oligos identified two functional domains within the pRNA, one for procapsid binding and one for DNA translocation. A series of deletion mutants of the pRNA distinguished the boundary of the pRNA functional domains, and revealed the minimum pRNA sequence requirement for specific procapsid binding and DNA packaging. The results of the deletion mutagenesis analysis clearly indicates that the 5′/3 ′ helical region of the pRNA plays a crucial role in DNA translocation but is dispensable for procapsid binding. By UV crosslinking, the pRNA was found to bind specifically to the connector and not to the capsid or scaffolding proteins of the procapsid. The structure of the pRNA was also probed by site-specific photoaffinity crosslinking. Results of structure probing revealed bases involved in both inter- and intramolecular interactions. Inter-pRNA crosslink dimers retained full activity in phi29 procapsid binding and genomic DNA translocation, indicating that the crosslink distance constraints identified in dimer formation reflect the native pRNA complex. Crosslinked dimers either containing or not containing the interlocking loops for hexamer formation bound procapsid equally well, however, only the one containing the interlocking loops for programmed hexamer formation was active in phi29 DNA packaging. Intramolecular crosslinks of three cp-pRNAs revealed bases within the pRNA that are proximate. Purified intramolecular crosslinks retained substantial procapsid binding activity, however, these crosslinks exhibited reduced DNA packaging activity. These results suggesting that the pRNA undergoes a conformational change during DNA packaging that is not necessary for procapsid binding. Crosslink distance constraints obtained from both inter- and intramolecular crosslinks provide a background for future modeling of the pRNA tertiary structure.
Degree
Ph.D.
Advisors
Guo, Purdue University.
Subject Area
Molecular biology
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