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Early T7 gene expression

Rates of RNA synthesis and degradation, protein kinase dependent termination of transcription, and efficiency of translation

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Summary

The mode of transcription of early T7 genes starting from one promotor region and generating a unique polycistronic RNA species suggests the appearance of equimolar amounts of the monocistronic species after RNA processing. Rate measurements revealed, however, a disproportion in the generation of the individual early RNA species. The rate of appearance of the promotor-proximal M gene message (nomenclature see in Table 1) is 4–5x the rate of appearance of all other species. This rate pattern is caused by termination behind the M gene because i) the rate of RNA degradation is fairly similar for most RNA species and ii) termination behind the M gene is released in a T7 mutant lacking protein kinase or in wild type infections in the absence of protein synthesis. Then, the RNA species are produced in equimolar amounts.

The rate of degradation is similar for all early RNA species except for the protein kinase message. As measured by two independent methods, the physical halflives of M, POL, 1.1, and LIG (nomenclature see Table 1) message were 7–8 min (30°), while KIN RNA was degraded with a halflife of 4 min. The functional halflives were around 50% of the physical halflives. There is apparently no relationship between size of RNA and halflife, and the data suggest specific signals on each RNA which determine the rate of degradation.

The monocistronic RNA species are utilized with different rates in translation. The M gene is not only transcribed more often, it is also translated with highest efficiency. The in vivo translation of the POL gene message occurred with the lowest rate.

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Abbreviations

tris, SDS, EDTA, TEMED, PPO:

DMSO see Materials

trp:

tryptophane biosynthetic operon

KIN:

T7 protein kinase

POL:

T7 RNA polymerase

LIG:

T7 DNA ligase

M:

membrane altering protein

T7:

protein required to overcome host restriction, identical with “0.3” protein according to the nomenclature of Simon and Studier (1973)

A600 :

optimal density at 600 nm

TB:

tryptone broth

RNase:

ribonuclease

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Author notes

  1. Helmut Ponta, Hans-Jobst Rahmsdorf & Peter Herrlich

    Present address: Universität Innsbruck, Institut für Biochemie, Naturwissenschaftliche Fakultät, Innsbruck, Germany

Authors and Affiliations

  1. Max-Planck-Institut für Molekulare Genetik, Berlin Dahlem, Germany

    Mei-li Pfennig-Yeh, Helmut Ponta, Hans-Jobst Rahmsdorf & Peter Herrlich

  2. Institut für Genetik der Univ. Karlsruhe, Postfach 3640, D-7500, Karlsruhe 1, Federal Republic of Germany

    Monica Hirsch-Kauffmann & Manfred Schweiger

Authors
  1. Mei-li Pfennig-Yeh
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  2. Helmut Ponta
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  3. Monica Hirsch-Kauffmann
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  4. Hans-Jobst Rahmsdorf
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  5. Peter Herrlich
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  6. Manfred Schweiger
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Communicated by W. Arber

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Pfennig-Yeh, Ml., Ponta, H., Hirsch-Kauffmann, M. et al. Early T7 gene expression. Molec. Gen. Genet. 166, 127–140 (1978). https://doi.org/10.1007/BF00285915

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