Structure of RNA and DNA chains in paused transcription complexes containing Escherichia coli RNA polymerase

Abstract

RNA polymerases pause conspicuously at certain positions on a DNA template. At the well-studied pause sites in the attenuation control regions that precede the trp and his operons, both formation of secondary structure in the nascent transcript and the DNA sequence immediately downstream contribute to pausing. The mechanisms of these effects are unknown. We report here studies on the structure of the RNA and DNA strands in purified trp and his paused transcription complexes in comparison to ten elongation complexes halted by nucleoside triphosphate deprivation. A 14 to 22 nucleotide region of the DNA strands was accessible to modification by KMnO4 or diethylpyrocarbonate in both the paused and halted transcription complexes. However, the region in front of the nucleotide-addition site was reactive only in some halted complexes. In both types of complexes, approximately eight nucleotides on the template strand immediately preceding the 3' end were protected from modification. We also examined the sensitivity of the nascent transcript to RNase A and found that the 3'-proximal eight nucleotide region could be cleaved without complete loss of the potential for elongation. However, a model RNA:DNA hybrid designed to mimic a hybrid in the transcription complex could also be cleaved under similar conditions. Together, the results suggest that the 3'-proximal eight nucleotides of transcript may pair with the DNA template and that this structure is not disrupted by hairpin formation at a pause site. Rather, pausing may result from distinct interactions between RNA polymerase and both the pause RNA hairpin and the downstream DNA sequence.