Transcription termination at the attenuators of the trp operons of Escherichia coli and Salmonella typhimurium was studied in vitro using DNA restriction fragments as templates. Readthrough transcription beyond the terminators occurred with 5 and 30% efficiency, respectively, in E. coli and S. typhimurium. This difference is correlated with the stability of proposed secondary structures of the respective trp leader transcripts. Secondary structure analyses of the two leader transcripts revealed a well-conserved pattern of RNA base pairing. This and the possibility that trp leader RNA is translated suggest a model for regulation of transcription termination that is based on ribosome movement along the RNA and a shift between alternative RNA base-pairing configurations.The tryptophan (trp) operon of Escherichia coli has two transcription control sites, a promoter-operator (14) and an attenuator (4). At the attenuator, a site located in the transcribed leader segment of the operon, transcription is either terminated or allowed to continue into the structural genes of the operon (4). Transcription termination at the attenuator appears to be regulated in response to changes in the extent of charging of tRNATrP (5, 6). Salmonella typhimurium has a trp attenuator that functions much like the one in E. coil (F. Lee and C. Yanofsky, unpublished). The sequences of the leader-attenuator regions of the trp operons of both species have been determined (F. Lee, K. Bertrand, G. Bennett, and C. Yanofsky, unpublished.In this paper we report the results of in vitro transcription studies with restriction fragments of the tip operons of both organisms. We show that the tip leader transcripts have appreciable secondary structure. We suggest how this structure may play a role in regulating transcription termination at the attenuator.
MATERIALS AND METHODSRestriction fragments of the E. coli and S. typhimurium trp operons were derived from plasmids pVH153 (7) mM Tris, pH 7.9/10 mM MgCl2/0.1 mM EDTA, and RNase T1 was added to a final concentration of 10 units/ml. Incubations were carried out at 200. Samples were withdrawn at various times, chilled, and then diluted with the same buffer. Diethylpyrocarbonate was added to 1% before the samples were precipitated with ethanol in the presence of carrier tRNA. They were then dissolved in urea/dye solution and loaded on denaturing gels. RNA fragments were eluted from polyacrylamide gels, digested to completion with RNase T1, and fingerprinted (11).All procedures using recombinant DNA were performed in accordance with the National Institutes of Health Guidelines. RESULTS Transcription Termination on trp Operon Restriction Fragments. Previous studies in vitro established that when purified RNA polymerase transcribes the trp operon of E. coli, it generally terminates transcription after synthesizing the first 140 residues of trp RNA (12). The 3'-OH termini of the in vitro RNA transcripts are at about the same position as the termini of the in vivo transcripts (13). Thus, in vitro, RNA ...