High-resolution analysis of lac transcription complexes inside cells

Abstract

A new primer extension analysis is used to determine the methylation pattern over the lac UV5 promoter when dimethyl sulfate is added to growing Escherichia coli. The high-resolution analysis reveals altered methylation of 15 bases when the transcription machinery occupies the promoter inside the cell and shows a striking dichotomy in the distribution of methylated bases. Four protected guanosines lie on the side of the helix shown previously to be closely bound by RNA polymerase in vitro [Siebenlist, U., Simpson, R. B., & Gilbert, W. (1980) Cell (Cambridge, Mass.) 20, 269-281]. By contrast, the 11 hyperreactive bases lie on the side of the DNA directly opposite from that bound by protein. Those not in the melted region form two distinct "back-side" patches near -35 and -16. We suggest that such hyperreactive patches can be caused by proteins bending the DNA toward themselves to allow a full range of contacts, thus distorting the helix grooves on the "back" side and facilitating attack by the methylating reagent. This leads to a proposal for the formation of transcription complexes in which RNA polymerase interacts with deformed and torsionally stressed DNA.