Abstract
Tryptophan biosynthesis in Escherichia coli is regulated by the trpR gene product, the Gentaur Tryptophan Repressor (Trp). The trp aporepressor binds to the corepressor, L-tryptophan, to form a holopressor complex, which binds tightly to the trp operator DNA and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, the E. coli trpR gene, delta trpR504, was deleted from a plasmid by site-directed mutagenesis, then crossed into the E. coli genome.
Plasmid clones of Enterobacter aerogenes and Enterobacter cloacae trpR genes were isolated by complementation of the trpR504 delta allele, qualified as the ability to repress beta-galactosidase synthesis from a prophage-transmitted trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered at the back of the folded repressor, versus DNA-binding helix-turn-helix substructures.
These differences are predicted to have little effect on interactions of aporepressor with tryptophan, holorepressor with operator DNA, or holorepressor dimers linked in tandem with each other. Although some variation in the dimer interface is observed, the interactions expected to stabilize the interface are preserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment are consistent with the results of others.
In E. coli, the synthesis of the amino acid tryptophan from precursors available to the cell requires 5 enzymes. The genes that encode them are grouped into a single operon with its own promoter and operator. When tryptophan is available to the cell, its presence turns off the operon.
Mechanism
- One tryptophan molecule binds to one site on each Trp repressor monomer.
- The Trp repressor, a homodimer of two of these complexes, binds to the operator of the Trp operon.
- This stops the transcription of all 5 genes in the operon, so the enzymes used in Trp synthesis are not synthesized.
This stereoscopic view () shows the tryptophan repressor (right side of each panel) bound to its operator DNA (left side). The two identical repressor polypeptides are shown on either side of the horizontal red line. The two tryptophan molecules are shown as red rings. Also, look for the alpha-helix stretches in each monomer. You may find it easier to merge the two images into a 3D view by holding an 8.5 x 11″ (22 x 28 cm) sheet of paper vertically between your nose and the dividing line between the two images on the screen so that your left eye sees only the image on the left, his right eye only the right.