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Abstract
We have examined the effects of removing individual template nucleosides on promoter escape by Escherichia coli RNA polymerase in vitro. The ability of DNA templates containing random single nucleoside gaps generated by hydroxyl radical treatment to support the production of stable ternary transcription complexes was analyzed. On two templates containing different promoter and initial transcribed regions, we found that removal of nucleosides on the template strand in the region from -13 to at least +8 relative to the transcription start site interfered with ternary complex formation. The downstream border of this region varied for the two templates, suggesting an effect of the specific nucleotide sequence on the stability of intermediates in the promoter escape process. On the nontemplate strand, removal of nucleosides in the vicinity of the -10 consensus promoter element interfered with escape, whereas removal of nucleosides in the vicinity of the transcription start site actually enhanced the yield of ternary complexes. On one template, removal of nucleosides in an A-tract containing region upstream of the promoter caused a significant decrease in promoter escape, consistent with previous suggestions that contacts between this region and the RNA polymerase play a role in promoter binding and/or initiation.
Highlights
We have examined the effects of removing individual template nucleosides on promoter escape by Escherichia coli RNA polymerase in vitro
On two templates containing different promoter and initial transcribed regions, we found that removal of nucleosides on the template strand in the region from ؊13 to at least ؉8 relative to the transcription start site interfered with ternary complex formation
Significant and periodic differences between the populations of gapped molecules found in the EPonuc band and the ternary complex band are observed for template 1 (Fig. 7), especially in the vicinity of an A-tract found at positions Ϫ53 to Ϫ56, where missing nucleosides severely interfere with promoter escape
Summary
The missing nucleoside technique [12] uses native gel electrophoresis to discriminate among a population of DNA molecules, which have been randomly gapped by treatment with hydroxyl radical, according to their ability to form a specific protein-DNA complex. Native gel electrophoresis can resolve ternary complexes of RNAP, DNA, and RNA from binary complexes containing only enzyme and DNA [1, 13, 14], and ternary complexes themselves can exhibit varied mobilities based on a number of contributing factors that may include polymerase subunit composition or conformation, degree and position of DNA bending, and length of RNA product. We decided that these properties presented a rich opportunity to dissect the roles of various template nucleosides in the events leading from promoter binding to the formation of a productively elongating transcription complex
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