PMID:2670923
Levin, HL, Park, K and Schachman, HK (1989) Attenuation in the regulation of the pyrBI operon in Escherichia coli. In vivo studies of transcriptional termination.J. Biol. Chem. 264:14638-45
| Abstract | The attenuation model for transcriptional regulation of the Escherichia coli pyrBI operon is based on the assumption that transcription terminates upstream of the structural genes at a rho-independent terminator when cells contain high levels of UTP. When, however, the cells are limited for pyrimidines, the presence of ribosomes translating the short leader peptide is presumed to cause an alteration in the secondary structure of the terminator in a way that allows RNA polymerase to transcribe the entire operon. These two premises of transcriptional regulation were tested by using exonuclease protection assays to map the 3' ends of transcripts extracted from cells containing either ample or depleted concentrations of pyrimidines. The results support the model since 99% of the pyrBI transcripts terminated at the (G + C)-rich region of dyad symmetry upstream of the structural genes when cells were grown in excess uracil. In addition, a significant portion (36%) of the pyrBI transcripts extracted from cells containing reduced pyrimidine concentrations extended past the dyad into the structural genes. This observation correlated with the amounts of aspartate transcarbamoylase synthesized in cells under the various conditions. The mapping technique was also used to determine the position of the 5' ends of the transcripts to measure contributions of two potential start sites (P1 and P2) to the pool of pyrBI transcripts. The results show that under all conditions no more than 3% of the total transcripts had 5' ends corresponding to the upstream promoter, P1. In cells lacking P1 virtually all transcripts from P2 terminated at the (G + C)-rich hairpin when the cellular level of pyrimidines was high. Conversely 57% of the transcripts extended past the terminator when cells were grown in UMP. The S1 nuclease technique also provided a measure of the steady state level of transcripts originating at P2. In cells depleted of pyrimidines there was a 5-10-fold increase in these transcripts depending on the number of copies of pyrBI. This increase, which is independent of attenuation, is caused by a different regulatory mechanism which as yet has not been identified. |
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