CONFIRMATION OF THE HELICAL STRUCTURE OF THE 5' 3'-TERMINI OF THE ESSENTIAL DNA PACKAGING PRNA OF PHAGE PHI-29/

Bacteriophage phi 29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during viral assemb ly. An intriguing feature of phi 29 is the presence of a 120-base viru s-encoded RNA (pRNA) that is indispensable for DNA packaging. Phylogen etic comparison of similar RNAs in numerous phages has revealed that t he secondary structure of the pRNA is well conserved. Computer analysi s predicts the presence of an extensive segment of helix with three si ngle-base bulges generated by the pairing of the 5' and 3' ends. The d esire to understand the role played by the pRNA in DNA packaging has l ed to a mutational analysis of the 5'-/3'-terminal region, which is be lieved to be important in DNA translocation. Deletion of 3 bases from the 3' end of the RNA, shortening the pRNA from 120 to 117 bases, was tolerated without loss of activity, but additional deletion of the bas e 117 resulted in 100-fold less activity, and a 115-base pRNA was virt ually nonfunctional. Additionally, the three unpaired one-base bulges within the helical stretches of the paired proximate ends were nonesse ntial for pRNA activity, as demonstrated by deletion of the bulge indi vidually. An extensive series of helix disruptions by single- and mult iple-base substitution almost invariably led to the loss of DNA packag ing activity. Additional mutations that restored predicted base pairin gs rescued pRNA activity. This second site suppression confirmed that the 5'- and 3'-end region was paired and was indeed a helical stretch. The secondary structure was of greater importance than the primary se quence, with the exception of the requirement of an adenine at either the third or fourth position. The specific requirement of an adenine i n phi 29 pRNA at this position, as well as conservation of this positi on in other phage pRNAs, implicates that this base may play a special role in either the DNA-packaging reaction or the maintenance of the pR NA tertiary structure.