RNA-protein interactions in a Nus A-containing Escherichia coli transcription complex paused at an RNA hairpin.

We have isolated Escherichia coli transcription complexes, paused in the presence and absence of Nus A, which contain RNA substituted at every UMP residue with a photocrosslinking nucleotide analog. The pause site is immediately downstream from an RNA stem-loop structure, and although pausing occurs in the absence of Nus A, it is substantially enhanced in the presence of Nus A. We have analyzed the secondary structure of this RNA and show that the analog does not interfere with the formation of the normal stem-loop structures. Additionally, the analog substrate does not alter transcriptional pausing, in the presence or absence of Nus A, indicating that Nus A recognition of the transcription complex is not affected by the presence of the crosslinking groups in the RNA. Ribonuclease digestion of the RNA in paused complexes identifies two accessible regions, two nucleotides in the loop and one near the base of the upstream side of the stem-loop. Cleavage at one loop nucleotide is enhanced by Nus A, while the nucleotide near the base of the stem-loop is partially protected. Upon irradiation of the transcription complex, Nus A is not photoaffinity labeled by the RNA, even at a high molar ration to RNA polymerase (250:1). Both the beta and beta' subunits are labeled, however, indicating that the putative stem-loop binding domain on the core polymerase involves both subunits. Because the nucleotide protected from ribonuclease by Nus A is very near two analogs, yet Nus A is not crosslinked to the RNA, it is unlikely that Nus A could be protecting this position through direct contact. Furthermore, analog is substituted at positions in both the loop and at several positions in the stem, and again, no crosslinking to Nus A is observed. We conclude that enhancement of pausing by Nus A probably does not require direct interaction with the bases in the RNA stem-loop.