Regulation of the gltBDF operon of Escherichia coli: how is a leucine-insensitive operon regulated by the leucine-responsive regulatory protein?
The regulon controlled by the leucine-responsive regulatory protein (Lrp) of Escherichia coli consists of over 40 genes and proteins whose expression is regulated, either positively or negatively, by Lrp. The gltBDF operon, encoding glutamate synthase, was originally identified as a member of the Lrp regulon through a two-dimensional electrophoretic analysis of polypeptides from isogenic strains containing or lacking a functional Lrp protein. We have now demonstrated that Lrp regulates the transcription of gltBDF::lacZ operon fusions. Relative to expression in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium, gltBDF::lacZ expression in an lrp+ strain is repressed 2.2-fold in the presence of 10 mM exogenous leucine and 16-fold in Luria broth. Repression of gltBDF::lacZ expression by leucine or Luria broth is not seen for an isogenic strain containing a Tn10 insertion in lrp, and expression of gltBDF::lacZ is 44-fold lower than in the lrp+ strain when both are grown in glucose minimal MOPS medium. Lrp binds specifically to DNA fragments containing the gltBDF promoter region. Saturating levels of leucine do not abolish binding of Lrp upstream of gltBDF but merely increase its apparent dissociation constant from 2.0 to 6.9 nM. Electrophoretic analysis of the Lrp regulon established that target proteins differ greatly in the degree to which the effect of Lrp on their expression is antagonized by leucine. On the basis of our present results, we present a model for positive regulation of target genes by Lrp. Insensitivity to leucine would be expected when the effective intracellular concentration of Lrp is high relative to the affinity of Lrp binding sites required for transcription of the target gene. At lower concentrations of Lrp, transcription of the target gene should be sensitive to leucine. This model suggests that regulation of the concentration of active Lrp is critical to control of the Lrp regulon.
Ernsting
B R
BR
Biophysics Research Division, University of Michigan, Ann Arbor 48109.
Denninger
J W
JW
Blumenthal
R M
RM
Matthews
R G
RG
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000378:metabolism / Q000494:pharmacology*; D051756:Leucine-Responsive Regulatory Protein; D008969:Molecular Sequence Data; D009876:Operon / Q000187:drug effects; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015183:Restriction Mapping; D014157:Transcription Factors; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein; D001616:beta-Galactosidase; D005974:Glutamate-Ammonia Ligase; D007930:Leucine