An electron microscopic comparison of transcription on linear and superhelical DNA

Abstract

Electron microscopy of nascent RNA chains has been used to localize promoters on linear and negatively superhelical λ DNA. Transcription was performed in vitro using Escherichia coli RNA polymerase. RNA from four promoters was seen on linear λ DNA; these include the λ PL and PR promoters, which are the main “early” λ promoters in vivo, and two promoters within the b2 region. In order to orient the circular DNA for electron microscopy, a restriction enzyme isolated from Streptomyces albus G. (Sal I) was used to cleave the DNA at unique points. The Sal I cleavage sites on λ DNA have been determined to be at 67.3% and 68.3% on the linear map. Through visualization of nascent RNA transcribed from superhelical λ DNA it is concluded that: (a) transcription increases from PL and PR with a particularly large increase from PL; (b) there is activation of transcription from promoters that are not used on the linear DNA and that coincide with the areas of λ which are most readily denatured and which possess the highest A + T content. The activation of such regions and the increased efficiency of the promoters used on linear DNA are discussed in terms of the energetics of unwinding a negatively superhelical DNA by a ligand such as RNA polymerase. The association of A + T-rich regions with promoters and the possible role of superhelicity in transcriptional activation in vivo are discussed.