Mutational analysis of the primer RNA template region in the replication origin ( oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase

The primase-dependent phage G4 origin of complementary DNA strand synthesis ( G4 ori c ) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA). Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4 ori c pRNA template region. Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4 ori c activity. However, functional G4 ori c activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I. Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4 ori c functional activity. These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4 ori c function.