Molecular interaction between simian virus 40 DNA and Escherichia coli RNA polymerase. Mapping of the initiation sites on supercoiled and linear DNA

Abstract

A detailed map of the initiation sites recognized by the Escherichia coli RNA polymerase (holoenzyme) on simian virus 40 (SV40) DNA has been constructed. An approach is described in order to compare the sites recognized on supercoiled as well as relaxed linear DNA. For localization of the initiation sites on supercoiled DNA, RNA polymerase-SV40 DNA Form I binary complexes are stabilized by incubation with three ribonucleoside triphosphates. The stable initiation complexes are cleaved with single cut restriction endonucleases in order to generate termination sites at precisely defined locations. Elongation of the RNA chains up to the cleavage site produces a defined set of discrete RNA species, whose size can be determined with accuracy by agarose gel electrophoresis following glyoxal denaturation. Each transcript is oriented by following two different experimental approaches. 1) The DNA of an SV40 deletion mutant is used as a template in comparison with that of wild type SV40; and 2) the RNAs synthesized after cleavage with different single cut restriction endonucleases are compared. Determination of the length and polarity of these RNAs allows us to accurately position the initiation sites on the physical map of the SV40 genome. A similar analysis was conducted for the mapping of the initiation sites recognized on precut linear SV40 DNA. Comparison of the two templates (supercoiled and linear) reveals that modification of the DNA conformation affects selection by the enzyme of certain promoters. Although most of the promoters are common to both templates, four are specifically recognized on superhelical DNA, while two others are used exclusively on linear SV40 DNA.