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In Escherichia coli, one of the two genes encoding lysyl-tRNA synthetase, lysU, belongs to the regulon controlled by the leucine-responsive regulatory protein (Lrp). To map the site of Lrp action, mutants escaping regulation in rich medium were generated through random mutagenesis of the lysU promoter region. The mutations showed parallel effects on the strength of Lrp-DNA association, as measured in vitro by gel retardation experiments, and on the degree of repression of lysU expression by Lrp in vivo. In addition, DNase I and hydroxyl radical footprinting experiments indicated that several Lrp molecules bind to a DNA region of over 110 bp in a highly cooperative manner. This region, which encompasses the -35 box of the lysU promoter, was the target of all the mutations affecting the strength of the Lrp-DNA association. These mutations are frequently located in short A + T-rich runs distributed along the Lrp binding region with a periodicity of one helix turn. Because we could find such a regular alternance of A + T runs upstream of several other Lrp-regulated genes, we suggest that this pattern is one feature indicative of the binding of Lrp.

Using random Tn10 insertion mutagenesis, we isolated an Escherichia coli mutant strain affected in the regulation of lysU, the gene encoding the inducible form of lysyl-tRNA synthetase. The transposon giving rise to the altered expression of lysU was found inserted within lrp. The latter gene codes for the leucine-responsive regulatory protein (Lrp) which mediates a global response of the bacterium to leucine. An involvement of Lrp in the regulation of lysU was searched for by using a lysU-lacZ operon fusion. The following conclusions were reached: (i) inactivation of lrp causes an increased activity of the lysU promoter, whatever the growth conditions assayed, (ii) insertion of a wild-type lrp gene into a multi-copy plasmid significantly reduces lysU expression, and (iii) sensitivity of the lysU promoter to the presence of leucine in the growth medium is abolished in the lrp context.

We have constructed a series of 44 Escherichia coli strains in which the chromosomal region corresponding to the Ribosome Binding Site (RBS) of the lacZ gene, has been replaced by small DNA fragments harboring either RBSs from other genes, or artificial RBSs. The beta-galactosidase expression from these strains ranges from 1 to 130 per cent of that of the parental strain. Using this collection, we demonstrate here that strain-to-strain variations in expression are paralleled by nearly equivalent variations in lacZ mRNA content. We propose that, in this system, polarity and mRNA stability are tightly coupled to translation initiation, so that changes in RBS efficiency are detected mainly as changes in mRNA concentration rather than in the spacing between translating ribosomes. In addition, we show that the mRNA sequence immediately downstream from the initiator codon influences per se the lifetime of the lacZ mRNA. We discuss the mechanism of the interdependence between translation, transcription and degradation in this gene, and speculate about the general role of this interdependence in determining the expression of bacterial genes.