S1 Mapping to Locate 5’ and 3’ Ends of Transcripts

S1 Mapping to Locate 5’ and 3’ Ends of Transcripts

Early in the investigation of a gene and its biological action, it is helpful to learn its transcription start and stop points. One simple method for doing this is S1 mapping, which was developed by Berk and Sharp. S1 is the name of a nuclease that digests single-stranded RNA and DNA. S1 mapping shows on the DNA the endpoints of homology of RNA mole-cules. That is, the method can map the 5’ and 3’ ends of the RNA. Consider locating the 5’ end of a species of RNA present in cells. Messenger RNA is isolated from the organism, freed of contaminating protein and DNA, then hybridized to end-labeled, single-stranded DNA that covers the region of the 5’ end. After hybridization, the remaining single-stranded RNA and DNA tails are removed by digestion with S1 nuclease. The exact size of the DNA that has been protected from nuclease digestion can then be determined by electrophoresis on a DNA sequencing gel (Fig. 5.10). This size gives the distance from the labeled end of the DNA molecule to the point corresponding to the 5’ end of the RNA molecule.

Figure 5.10 S1 mapping to locatethe 5’ transcription start site. RNA extracted from cells is hybridized to a radioactive fragment covering the transcription start region, then digested with S1 nuclease. The de-natured complex is subjected to electrophoresis alongside size standards.