Distinct functional contributions of three potential secondary structures in the phage G4 origin of complementary DNA strand synthesis

Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4ori_c, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA. In a previous publication [Sakai et al., Gene 71 (1988) 323-330], we suggested that base pairing between the loops of stem-loops I and II, and/or II and III, might play a role in G4ori_c function. To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing. These mutations, however, did not seriously affect G4ori_c activity. This indicates that base pairing between the loops is not essential for G4ori_c functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4ori_c activity. To complete an analysis of the effects of altering the structure of the G4ori_c stem-loops, insertions were made into stem-loop III. In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4ori_c activity.