Summary
A novel element which relaxes stringent control when carried on a recombinant plasmid, pMY3, has been identified. The locus responsible is the Su+7 allele of the tRNATrp gene, for which the plasmid was originally selected.
Plasmids were constructed that were isogenic except at the tRNATrp locus. Those on which the Su+7 allele was efficiently transcribed conferred a relaxed phenotype on their hosts. Those which expressed the wild type (Su-7) gene did not. When spontaneous mutations of the tRNATrp genes to Su+7 on the latter plasmids were examined, these had gained the ability to relax their hosts.
Mutations on pMY3 that were selected for their loss of the relaxing effect also possessed reduced levels of Su+7 tRNA.
The cloned fragment in pMY3 carries a single copy of the tRNAAsp and Su+7 tRNA genes, which depend on a plasmid promoter for their expression. When the lac UV5 promoter is placed in tandem to the tRNA as in pMY204, Su+7 tRNA transcription is elevated and host cells are relaxed. When a gene which overproduces the lac repressor is introduced into cells carrying pMY204, synthesis of the tRNAs is dramatically reduced and stringent control of stable RNA synthesis is regained. That regained control can be overcome by IPTG, which relaxes the otherwise stringent plasmid-carrying cell.
The relaxation conferred by these plasmids correlates directly with the level of mature Su+7 tRNA present in the cells. This level must approach that of wild-type tRNATrp before relaxation ensues, though suppressor phenotype is evident at much lower tRNA levels.
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Abbreviations
- mD:
-
megadaltons
- MSI:
-
ppGpp
- MSII:
-
pppGpp
- IPTG:
-
Isopropyl-β-D-thiogalactopyranoside
- TCA:
-
Trichloracetic acid
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Communicated by L. Lerman
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Breeden, L., Yarus, M. & Cline, S. A cloned suppressor tRNA gene relaxes stringent control. Molec. Gen. Genet. 179, 125–133 (1980). https://doi.org/10.1007/BF00268454
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DOI: https://doi.org/10.1007/BF00268454