The Escherichia coli K-12 metJ193 allele contains a point mutation which alters the hydrophobic pocket responsible for in vitro binding of S-adenosylmethionine: effects on cell growth and induction of met regulon expression.
The metJ193 allele encodes one of two identified temperature-sensitive Escherichia coli K-12 met repressors. The nucleotide sequence of the metJ193 allele was determined. The point mutation was a T to A transversion at base 170 of the metJ193 open reading frame and resulted in the substitution of leucine by glutamine at the 56th amino acid residue of the MetJ193 protein. The mutational lesion altered the hydrophobic pocket responsible for in vitro binding of the corepressor S-adenosylmethionine by wild-type MetJ. MetJ193 protein formed at the permissive temperature (28 degrees C) allowed slow derepression of met regulon expression when cultures were shifted to the nonpermissive temperature (34 degrees C). When 28 degrees C cultures of strains bearing two metJ193 alleles were transferred from methionine-containing medium to minimal medium, derepression of met regulon expression did not occur quickly enough to avoid a lag in growth due to the methionine deprivation. The inability of the MetJ193 protein to easily accomplish transition between apo- and active-repressor conformations was also demonstrated by using a maxicell system to study expression of a plasmid-borne copy of the E. coli metF transcription unit. These results confirm the importance of the leucine 56 residue for the structure and function in vivo of the wild-type MetJ protein.
Collier
C D
CD
Department of Microbiology, Miami University, Oxford, Ohio 45056.
Johnson
J R
JR
D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator; D005838:Genotype; D005973:Glutamine; D007700:Kinetics; D007930:Leucine; D008190:Lyases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012097:Repressor Proteins / Q000235:genetics*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C064780:MetJ193 protein, E coli; D012097:Repressor Proteins; D014157:Transcription Factors; D005973:Glutamine; D012436:S-Adenosylmethionine; C022696:O-succinylhomoserine (thiol)-lyase; D008190:Lyases; D019757:Carbon-Oxygen Lyases; C019965:cystathionine beta-lyase; D007930:Leucine