CIRCULARLY PERMUTED VIRAL PRNA ACTIVE AND SPECIFIC IN THE PACKAGING BACTERIOPHAGE-PHI-29 DNA

A viral-encoded 120-base pRNA has been shown to have an essential role in the packaging of bacteriophage phi 29 DNA. The finding that both t he 5'- and 3'-termini of the pRNA are proximate and crucial for biolog ical function (C. Zhang, C. Lee, and P. Guo, 1994, Virology, 201, 77-8 5) prompted investigation of the activity of circularly permuted pRNAs (cpRNA) and of the expandability and essentiality of bases extending from the termini. A 117-base pRNA with a deletion of three bases downs tream of the proximal terminus was active in DNA packaging. Concatemer ic DNAs containing two tandem pRNA genes separated by a short or a lon g loop sequence were constructed. The cpRNAs from these DNA templates were transcribed in vitro and shown to be active in phi 29 DNA packagi ng, with activity comparable to the parental (noncircularly permuted) pRNA, indicating that neither of the loops tested affected the activit y and folding of the cpRNA. As few as four bases were sufficient to se rve as a loop for the terminal 180 degrees turn, and a loop as long as 27 bases did not affect the cpRNA structure and function. Eight cpRNA s were constructed to assess the effect of openings within the wild-ty pe pRNA structure. Opening of the bulge at residue 38 did not affect c pRNA activity, but opening the bulge at residue 55 greatly reduced it. Although the sequence of the 5',3'-terminal loop was not important fo r the folding and activity of the cpRNA, the activities of cpRNAs with openings at individual bulges or hairpins were different, indicating that each region plays a different role in pRNA folding and function. Our results indicate that it is possible to generate active circularly permuted pRNA by assigning internal sites of the pRNA as new 3'- and 5'-termini. The creation of new variable ends makes the labeling of in ternal bases of the pRNA molecule possible and will facilitate the ana lysis of pRNA secondary and tertiary structure. (C) 1995 Academic Pres s, Inc.