USE OF CIRCULAR PERMUTATION TO ASSESS 6 BULGES AND 4 LOOPS OF DNA-PACKAGING PRNA OF BACTERIOPHAGE PHI-29

A 120-base phage phi 29 encoded RNA (pRNA) has a novel role in DNA pac kaging. This pRNA possesses five single-base bulges, one three-base bu lge, one bifurcation bulge, one bulge loop, and two stem loops. Circul arly permuted pRNAs (cpRNA) were constructed to examine the function o f these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge co uld be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C(18)C(19)A(20)) bulge was dis pensable for procapsid binding, but was indispensable for DNA packagin g. The secondary structure around this CCA bulge and the phylogenetica lly conserved bases within or around it were investigated. Bases A(14) C(15)U(16) were confirmed, by compensatory modification, to pair with U(103)G(102)A(101) A(gg) was needed only to allow the proper folding o f CCA bulge in the appropriate sequence order and distance constraints . Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was es sential for procapsid binding, DNA packaging, and phage assembly. Disr uption of the middle of any one of the loops resulted in dramatic redu ctions in procapsid binding, subsequent DNA packaging, and phage assem bly activities. However, disruption of the loops at sequences that wer e close to double-stranded regions of the RNA did not interfere with p RNA activity significantly. Our results suggest that double-stranded h elical regions near these loops were most likely not involved in inter actions with components of the DNA-packaging machinery. instead, these regions appear to be merely present to serve as a scaffolding to disp lay the single-stranded loops that are important for pRNA tertiary str ucture or for interaction with the procapsid or other packaging compon ents.