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The specificity of transcription in vitro of the tryptophan operon of Escherichia coli

II. The effect of Rho factor

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Summary

We have studied the effect of purified Rho factor on the synthesis in vitro of tryptophan (trp) mRNA. The system used for transcription consists of purified RNA polymerase from Escherichia coli, DNA isolated from a trp transducing strain ofΦ80 and termination factor Rho. A general characterization of this system showed that in buffers of high ionic strength (0.15M KCl) Rho did not interfere with the dissociation of the transcription complex after a transcription cycle. This indicates that reinitiation of trp mRNA synthesis occurs, a conclusion which is supported by the kinetics of the synthesis of this RNA.

Results of DNA-RNA hybridization experiments with the in vitro synthesized trp mRNA and λ trp DNA containing different sections of the trp operon showed that at low concentrations of Rho (below 0.6 μg/ml) the promotor-proximal and the promotor-distal rp genes are transcribed with nearly equal efficiency. At high concentrations of Rho (6 μg/ml), however, more than 90% of the trp mRNA originates from the promotor-proximal genes. Analysis by electrophoresis on polyacrylamide gels revealed that trp mRNA synthesized in the presence of high concentrations of Rho was homogeneous in size and that the length of the molecules was less than that of a complete polycistronic transcript. RNA synthesized in the presence of low concentrations of Rho was significantly longer and reached the size of a full-length trp mRNA. When RNA was synthesized without Rho most of the trp mRNA was much longer than the length of the trp operon.

From these results we conclude that under our conditions of synthesis Rho recognizes a termination signal at the end of the trp operon. At high concentrations Rho also causes termination of trp mRNA synthesis at a specific site within the operon.

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Authors and Affiliations

  1. Medical Biological Laboratory TNO, Rijswijk, The Netherlands

    Hans Pannekoek,  Bernard Perbal & Peter Pouwels

  2. Laboratory for Molecular Genetics, State University of Leiden, Leiden, The Netherlands

    Hans Pannekoek

  3. Département de Biologie Service de Biochemie, Centre d'Etudes Nucléaires de Saclay, Gif-sur-Yvette, France

    Bernard Perbal

Authors
  1. Hans Pannekoek
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  2. Bernard Perbal
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  3. Peter Pouwels
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Additional information

Communicated by E. Bautz

The first paper in this series is: Pannekoek, H., Pouwels, P. H. Molec. gen. Genet. 123, 159–172 (1973).

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Pannekoek, H., Perbal, B. & Pouwels, P. The specificity of transcription in vitro of the tryptophan operon of Escherichia coli. Molec. Gen. Genet. 132, 291–306 (1974). https://doi.org/10.1007/BF00268570

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  • DOI: https://doi.org/10.1007/BF00268570

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