Isolation and footprint analysis of the Escherichia coli thr leader paused transcription complex

The E. coli thr operon leader region contains a cluster of transcription pause sites upstream of the attenuator. In this report, we determine the exact sites of pausing and analyze the structure of the ternary complex by footprint techniques. Under synchronized transcription initiation conditions in vitro, three closely-spaced transcription pause sites were identified. These pause sites appeared downstream of the first region of dyad symmetry, which encodes an RNA hairpin in the transcript, and occurred at positions G¹¹², G¹¹⁴ and A¹¹⁶ of the thr leader RNA. The results showed that the half-life of the thr paused complexes at G¹¹² and G¹¹⁴ could be enhanced by limiting the concentration of the nucleoside triphosphate GTP in the transcription reactions. In addition, the half-life of the paused complexes was shown to increase in the presence of NusA protein. The thr leader complex that paused immediately before residues G¹¹² and G¹¹⁴ of the nascent transcript was isolated and its structure was analyzed with enzymatic and chemical cleavage reagents. The footprinting studies using DNase I showed that there were approximately 35 nucleotides on both strands of the DNA that were protected by RNA polymerase from DNase I cleavage. The DNA segment protected by RNA polymerase is approximately 19 nucleotides upstream and 14 nucleotides downstream of the pause sites. The results from hydroxyl radical footprints also showed a similar pattern of protection at the transcription pause sites. However, no significant differences in the footprinting patterns were observed in the presence or absence of NusA protein.