Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination.

Abstract

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 Salmonella typhimurium has a trp attenuator that functions much like the one in E. coil (F. Lee and C. Yanofsky, unpublished).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regions of the trp operons of both species have been determined (F. Lee, K. Bertrand, G. Bennett, and C. Yanofsky, unpublished.In this paper we report the results of in vitro transcription studies with restriction fragments of the tip operons of both organisms.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 Samples were withdrawn at various times, chilled, and then diluted with the same buffer.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 ...

Four RNA families with functional transient structures

“…Lee and Yanofsky (1977) concluded that the termination efficiency at the attenuator is correlated with the stability of an embedded secondary structure which is conserved between E. coli and Salmonnella typhimurium; the proposed structures agree with results of a partial RNase T1 digestion that exhibit digestion resistance in the distal portion of this transcript, i.e., where the structural features are located. 32 Thereafter, Oxender et al (1979) conducted structural probing with RNase T1 partial digestion followed by isolation of the comigrating pairing regions in a non-denaturing gel electrophoresis; the base-pairing regions were subsequently identified via denaturing gel electrophoresis and fingerprinting, based on which the two hairpins comprising the terminator structure were drawn. 33 Later on, the secondary structure of DNA template was ruled out as a contributor for this termination signal so the RNA structural features are the one causing the termination.…”

“…9,38,39 Indeed, the functional importance of the second hairpin for the transcriptional termination is illustrated by the reduced transcription termination frequency observed in experiments destabilising the central GCC pairing of this hairpin, such as by in vivo mutational analysis or in vitro substitution of G-C bond by I-C bond. 32,[40][41][42] Moreover, mutational analysis progressively disrupting the first hairpin still preserves the production of the attenuated transcript, suggesting that the second hairpin itself is sufficient for the termination. 9,42 Anti-terminator structure Anti-terminator structure disrupts the two terminator hairpins…”

“…10,18,30,31 Whether the ribosome stalls during translation and where this occurs is likely to influence this regulatory attenuation mechanism because it depends on the abundance of the corresponding loaded tRNA; given the trailing trp tandem observed in this trp operon, the trp codon is found to be the codon responsible for this. 10,32 Where the ribosome stalls dictates which alternative RNA structure the leader transcript forms: either the transcription termination hairpin forms or it is disrupted to permit transcriptional read-through. 30 In essence, the choice between terminator and anti-terminator structures bridges the communication between the translation of the leader peptide and the transcription of operon genes in exchanging the message for the abundance of trp.…”

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

“…1) (4,32,38). In principle, the attenuation mechanism controlling the ilv operon appears to be quite similar to that found to control operons involved in tryptophan (35), leucine (19), phenylalanine (61), histidine (3), and threonine (17) biosynthesis: the leader region specifies a short peptide containing a disproportionate frequency of the amino acids that are found to regulate the operon. Thus, the rate at which the leader can be translated serves to sense whether the regulatory amino acid is in short or ample supply.…”

Intrastrand Self-complementary Sequences in Bacillus subtilis DNA

“…. C'B'A' have been described in the leader sequences in both prokaryotes (Lee & Yanofsky, 1977) and animal viruses (Chow et al, 1977), at the ends of insertion elements (Ohtsubo & Ohtsubo, 1978) andin several transposable DNA segments carrying antibiotic resistance genes (Kleckner et al, 1975;Heffron et al, 1975). It is possible that many modes of recombination occur through self-complementary sequences or that they are involved in the regulation of transcription.…”