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Amber suppression relaxes stringent control by elongating stringent factor

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Summary

Efficient expression of an amber suppressing tRNA Su+7, relaxes E. coli's stringent response to amino acid starvation. This suppressor tRNA interferes with the accumulation of (p)ppGpp rather than the cell's ability to respond to it, and this appears to be independent of which amino acid is withdrawn.

Isogenic UAA- or UGA-reading derivatives of Su+7 do not relax their hosts, but all other UAG suppressors tested also show the control effect. In fact, the extent of relaxation induced by a given amber suppressor is directly proportional to its suppressor efficiency. Suppressor tRNAs do not directly effect relaxation because when Su+7 expression is induced with IPTG, it takes twice as long to achieve full relaxation as it takes to reach the maximum level of Su+7 accumulation. This suggests that the tRNA does not affect relaxation directly but rather causes the accumulation of a secondary effector.

The nature of this secondary effector was determined using antibodies to stringent factor. In Su+7-bearing cells, half of the stringent factor antigen migrates on SDS polyacrylamide gels as if it is about 30 amino acids longer than the wild type protein. The ratio of elongated to wild type stringent factor is directly correlated with the amber suppressor efficiency of the cell's resident Su+ tRNA. When half the cell's stringent factor is elongated, it can make half as much (p)ppGpp in response to amino acid starvation. When a second gene for stringent factor is introduced to these cells, the amount of wild type stringent factor is doubled and stringency is restored, confirming that the effect on the stringent factor gene product is sufficient to explain the tRNA effect on stringent control.

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Abbreviations

TCA:

Trichloroacetic acid

IPTG:

Isopropyl-β-D-thiogalactopyranoside

EMS:

Ethyl methane sulfonate

Kd:

Kilodalton

SDS:

Sodium dodecyl sulfate

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Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, 80309, Boulder, CO, USA

    Linda Breeden & Michael Yarus

Authors
  1. Linda Breeden
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  2. Michael Yarus
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Additional information

Communicated by G. O'Donovan

This work was taken from the doctoral thesis of L.B. submitted to the University of Colorado, 1981

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Breeden, L., Yarus, M. Amber suppression relaxes stringent control by elongating stringent factor. Molec Gen Genet 187, 254–264 (1982). https://doi.org/10.1007/BF00331127

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  • DOI: https://doi.org/10.1007/BF00331127

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