Structure and function studies of the bacteriophage phi29 pRNA required for DNA packaging

Mark D. Trottier, Purdue University

Abstract

Bacteriophage ø29 utilizes a viral-encoded RNA (pRNA) to insert its genomic DNA into the viral procapsid. Mutant pRNAs were able to efficiently inhibit viral assembly in vitro and completely inhibit assembly in vivo. The efficiency of inhibition was attributed to, one, that the pRNA contained two domains, one for procapsid binding and one for DNA translocation, which allowed construction of competitive RNA inhibitors of DNA packaging, and two, that multiple copies of the pRNA were required for each DNA packaging event. The ability of mutant pRNAs to efficiently inhibit in vitro ø29 assembly was used as a means to determine the stoichiometry of the pRNA. The probability of a procapsid binding a certain number of wild type and a certain number of mutant pRNAs was predicted, and this probability was used to predict the level of inhibition of ø29 assembly by mutant pRNA. Also, comparison of the slopes of curves of dilution factor versus yield of infectious virions in in vitro ø29 assembly for various ø29 assembly components was performed. The larger the stoichiometry of the component, the more dramatic the influence of the dilution factor on the reaction. The results of the stoichiometric analyses supported the conclusion that 5 to 6 pRNAs per procapsid were required for DNA packaging. The structure of the pRNA and pRNA mutants was also probed by chemical modification. Results of structure probing supported the predicted secondary structure of the pRNA. In the absence of Mg++, reduced modification of loops in the procapsid binding domain was observed, possibly accounting for the inability of the pRNA to bind procapsids under these conditions. Also, mutant pRNA P5/11, which had reduced procapsid binding affinity, had reduced modification in the lower loop. A mutant pRNA which had intact procapsid binding competence showed a modification pattern in the procapsid binding domain that was similar to that of wild type pRNA, further suggesting the importance of the loops of this region in procapsid. binding. Large areas of protection from chemical modification were observed when the pRNA was bound to procapsids, suggesting a large procapsid footprint.

Degree

Ph.D.

Advisors

Guo, Purdue University.

Subject Area

Molecular biology|Immunology|Microbiology|Biophysics

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