Summary
A recombinant plasmid, pMY3, was constructed in this laboratory to express the amber suppressor allele, Su+7, of the tRNATrp gene from E. coli (Yarus, 1979a). This plasmid also relaxes control of the synthesis of all stable RNA species in its host cell after amino acid deprivation. Guanosine penta and tetra-phosphate (MSII and MSI) concentrations are reduced to about one-half the levels achieved by starving the host cells carrying the cloning vehicle (pMB9) alone.
We now show that the relaxation conferred on cells carrying pMY3 can be overcome by at least three different missense mutations at the chromosomal spoT locus. In these stringent, plasmid-carrying strains, the ppGpp levels attained during starvation are equivalent to or higher than that of the host cell carrying the vehicle alone.
In vitro mutagenesis of the relaxing plasmid with EMS, followed by transformation and screening for plasmid-bearing stringent cells, yielded four stringent revertants of the relaxing locus. Cells carrying these mutants plasmids all have normal stringent responses to amino acid starvation, and again, elevate (p)ppGpp levels equal to or greater than 80% LS286 (pMB9) levels.
Despite pMY3′s modest effect on its host's MSI levels during the steady state of starvation, an obvious correlation exists between the concentration of that nucleotide and the host's ability to respond stringently. We therefore believe that the plasmid intervenes in MS metabolism. Measurements of the in vivo rates of decay of MSI and MSII after reversal of isoleucine starvation show that pMY3 has no effect on those reactions. The most likely mechanism of plasmid action is therefore inhibition of MS synthesis.
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Abbreviations
- MSI:
-
ppGpp
- MSII:
-
pppGpp
- EMS:
-
ethyl methane sulfonate
- TCA:
-
trichloroacetic acid
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Communicated by L. Lerman
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Breeden, L., Yarus, M. Mutations that overcome plasmid-mediated relaxation affect (p)ppGpp. Molec. Gen. Genet. 179, 119–124 (1980). https://doi.org/10.1007/BF00268453
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DOI: https://doi.org/10.1007/BF00268453