Trp Leader RNA Research Articles

Release of transcript and template during transcription termination at the trp operon attenuator.

We studied release of trp leader RNA and trp template DNA from RNA polymerase during transcription termination at the attenuator of the trp operon of Escherichia coli. Preliminary evidence had suggested that a stable ternary complex was formed at the trp attentuator. We observed that the complexes between RNA polymerase and trp leader RNA and the DNA template produced during transcription were labile at high salt concentrations and were undetectable when transcription was performed in the presence of heparin. These characteristics are atypical of the stable transcription termination complexes described by others (Richardson, J. P., and Conaway, R. (1980) Biochemistry 19, 4293-4299; Shigesada, K., and Wu, C. (1980) Nucleic Acids Res. 8, 3355-3369). We successfully reconstituted polymerase-trp leader RNA complexes in simple mixing experiments; these and other studies indicated that it is core polymerase that binds the leader transcript and the DNA template. In agreement with this conclusion, it was observed that sigma factor inhibited binding of RNA polymerase to the trp leader transcript and the DNA template and displaced leader RNA from RNA polymerase during transcription. It seems likely that small amounts of core polymerase present in the holoenzyme preparation, or generated during transcription, are responsible for the nonspecific binding of RNA transcript and DNA template. Our findings, therefore, suggest that the transcription termination event at the trp attenuator normally involves spontaneous dissociation of polymerase, template, and RNA transcript.

Studies on the involvement of the UGA readthrough process in the mechanism of attenuation of the tryptophan operon of Escherichia coli.

We asked if UGA suppression by charged tRNATrp, a process called UGA readthrough, is involved in the mechanism of attenuation of the tryptophan (trp) operon in Escherichia coli. For this purpose we used two mutations: strA(LD1) which causes restriction of UGA readthrough, and revA which partially overcomes the restriction of UGA readthrough caused by strA(LD1)(Engelberg-Kulka et al. 1982). trp attenuation was monitored by the regulation of the synthesis of the trp operon enzyme anthranilate synthetase (ASase) in trpR strains. We showed that the strA(LD1) mutation causes a significant increase in the level of synthesis of ASase in the presence of an excess of tryptophan, while the revA mutation reverses this effect, indicating that transcription termination at the trp attenuator site is relieved by restriction of UGA readthrough. Based on our results and the sequence data of the trp leader RNA of E. coli (Oxender et al. 1979), we offer a model for the involvement of the UGA readthrough process in trp attenuation. We suggest that the UGA readthrough process permits trp attenuation to respond to slight changes in the cellular concentration of charged tRNATrp.

Transcription termination at the tryptophan operon attenuator is decreased in vitro by an oligomer complementary to a segment of the leader transcript.

A DNA oligomer 15 nucleotides long was used to probe the involvement of RNA secondary structure in the control of transcription termination at the attenuator of the tryptophan (trp) operon of Escherichia coli. This 15-mer is perfectly complementary to a segment of trp RNA that is thought to play a role in regulation of attenuation. When added to an in vitro transcription reaction mixture containing wild-type E. coli or Salmonella typhimurium trp operon templates, the complementary 15-mer caused a 4-fold increase in read-through transcription. By contrast, the 15-mer did not affect attenuation when a mutant E. coli template was used that does not allow formation of a crucial RNA secondary structure. Control experiments established that oligomers that were not complementary to E. coli trp leader RNA did not affect attenuation and that the 15-mer did not reduce termination when the transcript lacked a complementary region. Other experiments established that the 15-mer did not increase read-through transcription by allowing RNA polymerase molecules that might have already stopped at the attenuator to resume transcription. These findings provide direct support for the view that alternate base-paired structures control transcription termination at the trp attenuator.

Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region.

The secondary structure of the terminated trp leader transcript from Escherichia coli was analyzed by RNase T1 partial digestion. Base-paired regions were recovered by nondenaturing gel electrophoresis and identified by denaturing gel electrophoresis and fingerprinting. The tandem tryptophan codons in the leader peptide coding region were found to be base paired with a more distal region of the transcript. This and other secondary structures that the trp leader RNA can form help explain the physiological response of the operon as well as the behavior of regulatory mutants.

Evidence for endonucleolytic cleavage at the 5′-proximal segment of the trp messenger RNA in Escherichia coli

The 5'-proximal trp leader RNA segment (about 5S) decays at 2 to 3 times slower rates than the distal trp mRNA sequence. This has been demonstrated by employing the deletion mutants which lack a large portion of the structural genes but retain the promoter-proximal region of the trp operon. Relative stability of the leader RNA is not merely due to the presence of an untranslatable region in the segment; the internal untranslatable segment of trp mRNA downstream from the nonsense alteration site of a double mutant trpAD28.trpE9758 decays as fast as the normal trp mRNA sequence. These results suggest that the trp mRNA is endonucleolytically cleaved to yield the small 5'-proximal leader RNA segment before the distal mRNA decays and that the leader RNA sequence is not subject to usual mode of mRNA decay in the 5' to 3' direction.

Translational control of transcription termination at the attenuator of the Escherichia coli tryptophan operon.

We have isolated two regulatory mutants altered in the leader region of the Escherichia coli tryptophan (trp) operon. In one mutant, trpL29, the AUG translation start codon for the trip leader peptide is replaced by AUA. The other mutant, trpL75, has a G leads to A change at residue 75, immediately after the UGA translation stop codon for the trp leader peptide. In vivo, trpL29 and trpL75 increase the efficiency of transcription termination at the trp attenuator 3- to 5-fold. trpL29 and trpL75 also fail to respond fully to tryptophan starvation and other conditions that normally relieve transcription termination at the trp attenuator. The trpL29 mutation, which presumably reduces synthesis of the trp leader peptide, is cis dominant. The effect of starvation for a number of the amino acids in the trp leader peptide was determined. Only starvation for tryptophan and arginine, amino acids that occur at residues 10, 11, and 12 of the 14-residue trp leader peptide, elicits relief of transcription termination. Our findings suggest that translation of trp leader RNA is involved in regulation of transcription termination at the attenuator. A model is discussed in which the location of the ribosome synthesizing the leader peptide is communicated to the RNA polymerase transcribing the leader region.

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

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 paring. 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 configuration.