Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd

The single-stranded DNA sequence d(GT(5)G(4)CT(4)C) occurs close to the ori gin of replication within the intergenic region of the viral strand of bact eriophage fd. The RNA analogue of this sequence r(GU(5)G(4)CU(4)C) forms pa rt of the untranslated leader sequence of the gene 2 mRNA and is specifical ly bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in th e control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filt ration and non-denaturing gel electrophoresis. The CD spectrum of the struc tured form is characteristic of parallel guanine tetraplexes. The structure d form of the DNA sequence melts at approx. 47 degrees C in the presence of Na+ ions but the structure is stabilized up to 75 degrees C in the presenc e of K+ ions. The RNA structure is more stable than the equivalent DNA stru cture (melting temperature approx. 62 degrees C), and its stability is furt her enhanced in the presence of K+ ions. Two of the central guanine residue s are fully protected from cleavage as determined by dimethyl sulphate prot ection experiments, whereas methylation interference studies show that meth ylation of any of the four central guanine residues inhibits structure form ation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in R NA this might be further stabilized by the presence of weaker uracil quarte ts.