SEQUENTIAL ACTION OF 6 VIRUS-ENCODED DNA-PACKAGING RNAS DURING PHAGE PHI-29 GENOMIC DNA TRANSLOCATION

A 120-base pRNA encoded by bacteriophage phi 29 has a novel and essent ial role in genomic DNA packaging, Six DNA-packaging RNAs (pRNAs) were bound to the sixfold symmetrical portal vertex of procapsids during t he DNA translocation process and left the procapsid after the DNA-pack aging reaction was completed, suggesting that the pRNA participated in the translocation of genomic DNA into procapsids. To further investig ate the mechanism of DNA packaging, it is crucial to determine whether these six pRNA molecules work as an integrated entity or each pRNA ac ts as a functional individual, If pRNAs work individually, then do the y work in sequence with communication or in random order without inter action? Results from compensation and complementation analysis did not support the integrated model, Computation of the probability of combi nation between wild-type and mutant pRNAs and experimental data of com petitive inhibition excluded the random model while favoring the propo sal that the six pRNAs functioned sequentially, Sequential action of t he pRNA also explains why the pRNA is so sensitive to mutation, since the effect of a pRNA mutation will be amplified by 6 orders of magnitu de after six consecutive steps, resulting in the observed complete los s of DNA-packaging activity caused by small alterations. When any one of the six pRNAs was replaced with an inactive one, complete blockage of DNA packaging resulted, strongly supporting the speculation that in dividual pRNAs, presumably together with other components such as the packaging ATPase gp16, take turns mediating successive steps of packag ing, Although the data provided here could not exclude the integrated model completely, there is no evidence so far to argue against the mod el of sequential action.