THE PROXIMATE 5' AND 3' ENDS OF THE 120-BASE VIRAL-RNA (PRNA) ARE CRUCIAL FOR THE PACKAGING OF BACTERIOPHAGE-PHI-29 DNA

In vitro mutagenesis was performed to identify the DNA packaging domai n of the 120-base pRNA essential and specific for DNA encapsidation by bacteriophage phi 29 of Bacillus subtilis. All deletions and mutation s targeted the 5' and 3' ends of the pRNA. DNA templates of a control or mutant pRNAs used for in vitro transcription with T7 RNA polymerase were generated by PCR. Fourteen mutant pRNA molecules were synthesize d from DNA templates either directly after PCR or after cloning the PC R fragments into the pCR II vector. Ten of the mutant pRNA species wer e inactive in packaging of the phi 29 genome. Mutation of base one at the 5' end did not affect the pRNA packaging activity. Mutation of the first two bases at the 5' end of the pRNA to noncomplementary bases i n the predicted RNA secondary structure (U-1 C-2/A(117)G(116) to G(1) G(2)/ A(117)G(116)) resulted in a pRNA with no detectable DNA-gp3 pack aging activity assayed by either sucrose gradient sedimentation or aga rose gel electrophoresis, and 10(5)-fold reduction in activity was fou nd when measured by plaque-forming units with a new highly sensitive a ssay system. Changing bases 116 and 117 so that they were complementar y to the mutated bases, 1 and 2, from the previous mutant (G(1) G(2)/A (117)G(116) to G(1) G(2)/C117C116) generated an RNA molecule with rest ored DNA packaging ability. Our results show that, although not essent ial for procapsid binding, both the 5' and 3' ends of the pRNA were pr oximate and crucial for phi 29 DNA packaging. (C) 1994 Academic Press, Inc.