Overexpression of the relA gene in Escherichia coli.
Intracellular levels of guanosine 3',5'-bispyrophosphate (ppGpp) governed by the relA gene are normally regulated by aminoacyl-tRNA availability for protein synthesis. An experimental system is described in which cellular levels of ppGpp are controlled instead by induction of plasmid pKK223-3 derivatives with the relA structural gene, or portions thereof, under control of the Ptac promoter. In amino acid-rich media, isopropyl-1-thio-beta-D-galactopyranoside induction of transcription of the wild type relA gene in pSM10 yields about a 100-fold overexpression of a metabolically stable, full length (743 amino acid) RelA protein to levels approximating the number of cellular ribosomes. This overexpression is accompanied by a roughly parallel and relC-dependent elevation of ppGpp levels. Induction of a relA gene deletion mutant in pSM11 containing 455 amino-terminal amino acids results in much lower levels of expression of a metabolically unstable 55-kDa protein and elevated ppGpp levels that are almost equivalent to induced pSM10 and are relC-independent. Induction of a larger deletion in pSM12 containing 331 amino-terminal amino acids does not provoke ppGpp accumulation. We are able to elicit high levels of ppGpp without changing nutritional abundance and without massive overexpression of the RelA protein by inducing the metabolically unstable, truncated RelA protein. We find the effects of elevated ppGpp levels to include a slowing of growth, an inhibition of stable RNA accumulation, an inhibition of cellular rrn P1 promoter activities as measured by primer extension, and changes in the pattern of gene expression viewed by two-dimensional electrophoresis of cellular proteins.
Schreiber
G
G
Department of Cellular Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Metzger
S
S
Aizenman
E
E
Roza
S
S
Cashel
M
M
Glaser
G
G
D001483:Base Sequence; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology; D014158:Transcription, Genetic
C028002:serine hydroxamate; D006159:Guanosine Tetraphosphate; D012694:Serine