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.
Highlights
The results indicate that structural features in SV40 DNA, as well as specific nucleotide The supercoiled DNAs (FormI) of the small papovaviruses sequences, are determinants in the choice of the promoters simian virus 40 (SV40)’ and polyoma have been extensively that will be utilized by the enzyme
Transcription of SV40 DNA Form I-SV40 DNA Form 1 was transcribed by the E. coli RNA polymerase for various lengths of time and theRNA was analyzed on 5 to 20%sucrose gradients or by gelelectrophoresis followingdenaturationwith glyoxal
Transcription of SV40 DNA Form III-Based on the above results, we predicted that when SV40 DNA cleaved at a specific site with a single cut restriction endonuclease was used as a template, termination of the growing RNA chains should occur at the ends of the linear molecule
Summary
PreformeEd. coli RNA polymerase-SV40 DNA tion of the lengthand polarity of these RNAs allows us Form I initiation complexes were cleaved with a single cut to accurately position the initiation sites on the physi- restriction endonuclease. These cleavages generate terminacal map of the SV40 genome. Binding sites of the Escherichia coli RNA polymerase on these two templates has been previously investigated either byretentionon nitrocellulose fiiters of the specific DNA fragments to which the enzyme is associated or by electron microscopic analysis of RNApolymerase-DNA complexes cleaved with a “single cut” restriction endonuclease(6, 9, 1012).
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