DNA structure equilibria in the human c-myc gene.
We have employed analytical S1 nuclease analysis to identify sites with altered DNA secondary structure in the human c-myc gene. We have mapped several sites of that kind in vitro at one-base resolution but have focused our attention on one particularly stable conformational isomer which occurs approximately 270 base pairs upstream from the preferred transcription origin. We have analyzed the kinetics of that conformational equilibrium as a function of supercoil density and enzyme concentration and find that DNA structure in this region is adequately modeled as a two-state equilibrium between an undistorted (S1 nuclease insensitive) and a distorted (S1-sensitive) state. We find that at fixed supercoil density, S1 nuclease cleavage at this DNA segment can be altered in vitro by a DNA sequence change as far away as 1500 bases. We also find that the S1 nuclease cleavage at this site can be dramatically enhanced by the binding of small RNA molecules. On the basis of an analysis of S1 cutting kinetics and an analysis of DNA sequence at the S1 cleavage site, we conclude that RNA may bind directly to DNA, thereby shifting the underlying conformational equilibrium. Together, these data suggest that as a class, short RNA molecules could serve as site-specific regulatory elements in the myc gene and elsewhere.
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004720:Endonucleases; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009857:Oncogenes; D010957:Plasmids; D006163:Ribonuclease T1; D015719:Single-Strand Specific DNA and RNA Endonucleases
D004247:DNA; D004720:Endonucleases; D006163:Ribonuclease T1; D015719:Single-Strand Specific DNA and RNA Endonucleases