Long range base pairing in bacteriophage φ X174 single-stranded DNA

Abstract

Standard formamide/cytochrome c spreading conditions, modified by the addition of 0·5 m-ammonium acetate, have been used to visualize reproducible structures resulting from long range base pairing in single-stranded DNA from bacteriophage φX174. The most highly folded circular viral DNA molecules appeared as three-lobed structures, and one or two of these lobes were also found in less folded molecules. The lobes defined three structural domains of characteristic size and morphology. The locations of these domains and seven of the stem and loop structures comprising them have been mapped relative to the molecular ends of denatured PstI-cleaved replicative form DNA. Their orientation relative to the φX174 genetic map and nucleotide sequence was determined using denatured HpaI-cleaved replicative form DNA. An examination of the nucleotide sequence in regions corresponding to the observed structures revealed potential base pairing with calculated negative free energies from −21 to −46 kcal/mol. The total length of the paired regions averaged 33 base-pairs, but the paired segments include no more than 11 consecutive base-pairs (including G.T pairs) interspersed with unpaired DNA of varying length. The observed structures and proposed base-paired sequences bring the 5′ and 3′ ends of linear φX174 DNA, cleaved at the origin of viral strand replication, close together which may facilitate their rejoining by φX174 gene A protein following DNA replication. In some but not all of the φX174 messenger RNA transcripts the ribosome binding sites for genes C and E may be involved in base pairing which might in part account for the relatively small quantities of their polypeptide products synthesized in vivo.