Nuclease cleavage of the upstream half of the nontemplate strand DNA in an Escherichia coli transcription elongation complex causes upstream translocation and transcriptional arrest

Abstract

We tested the susceptibility of nucleic acid strands in a halted transcription elongation complex to digestion by micrococcal nuclease (MN). The 16-nucleotide nascent RNA was protected within RNA polymerase. A 27-28- nucleotide template strand DNA fragment also was resistant to MN digestion. However, the upstream half of the nontemplate DNA within this region was digested rapidly by MN, suggesting that the nontemplate strand emerges from the RNA polymerase near the middle of the melted transcription bubble with the bases oriented away from the enzyme surface. MN cleavage of the exposed nontemplate DNA shifted polymerase backward, making it unable to extend the RNA chain. However, the MN-trimmed G16 complexes could be reactivated by GreB-stimulated cleavage of the nascent RNA. These results favor a model of transcriptional arrest involving upstream slippage of RNA polymerase along the RNA and DNA chains. They also suggest that the exposed segment of nontemplate DNA may directly or indirectly stabilize the lateral position of the transcription complex along the DNA.