Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions

Abstract

Five of the genes for the biosynthesis of isoleucine and valine form the ilvGMEDA operon of Escherichia coli K-12. Expression of the operon responds to changes in the availability of isoleucine, leucine, and valine (ILV). Addition of an excess of all three amino acids results in reduced expression of the operon, whereas limitation for one of the three amino acids causes an increase in expression. The operon is preceded by a leader-attenuator which clearly regulates the increased expression that occurs due to reduced aminoacylation of tRNA. To assess the factors that result in the reduced expression of this operon upon the addition of ILV, a series of plasmids were constructed in which the ilv regulatory region was fused to galK. In response to addition of the amino acids, expression of the galK gene fused to the leader-attenuator decreased five-to sevenfold, instead of the twofold observed for the chromosomal operon. A deletion analysis with these plasmids indicated that the ILV-specific decrease in expression required an intact leader-attenuator but not ilvGp2 or the DNA that precedes this promoter. This conclusion was supported by both Sl nuclease analysis of transcription initiation and determination of galK mRNA levels by RNA-RNA hybridization.Wild-type Escherichia coli K-12 can synthesize all its molecular components when grown on a minimal salts medium containing suitable carbon and nitrogen sources. During steady-state or exponential growth, the cells presumably synthesize the appropriate quantity of each metabolite so as to achieve optimal growth. This is facilitated by the ability of the cell to increase or decrease the synthesis of enzymes or alter the catalytic activity of specific enzymes in response to changes in the environment in which the cell is growing. For several amino acid-biosynthetic systems, the cell responds to an exogenous source of an amino acid (i.e., an excess) by decreasing (i.e., repressing) the expression of the relevant genes. Alternatively, if the amount of an amino acid becomes growth limiting (i.e., starvation), the bacteria will increase expression of the genes required for the biosynthesis of that amino acid.The trp operon is probably the most thoroughly studied set of genes for the biosynthesis of an amino acid. For the trp operon, the molecular mechanisms (34, 50) that result in the change in expression of this operon in response to either an excess or limitation for tryptophan are distinct. The trp repressor protein regulates the operon by mediating the initiation of transcription by RNA polymerase (RNAP) in response to an excess of free tryptophan within the cell.When growth is limited by tryptophan, increased expression of the operon occurs as a result of reduced aminoacylation of trp tRNA. This change in trp operon expression is mediated by the leader-attenuator. As described by Yanofsky et al. (50), the combination of the repressor-operator and the leader-attenuator results in a 500-to 600-fold range in expression of the trp operon.The ilvGMEDA operon encodes four of...

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

“…The same general conclusion comes from the more recent report of Lawther et al (33), who showed, in addition, that attenuation was dependent only on the intact leader region and not on either the ilv promoter itself or on those sequences upstream of the promoter that increased promoter strength.…”

Attenuation regulation of amino acid biosynthetic operons in proteobacteria: comparative genomics analysis

“…The ilvBN and ilvIH genes are transcribed as separate operons, whereas il-vGM is located within the ilvGMEDA operon. The il-vGMEDA and ilvBN operons are regulated by transcription attenuation, and the leader peptide reading frame of the attenuator contains regulatory codons for all three amino acids, isoleucine, leucine, and valine [9]. The leuABCD operon contains genes for the leucine biosynthesis and expression of this operon also is regulated by transcription attenuation [10].…”

Attenuation regulation of amino acid biosynthetic operons in proteobacteria: comparative genomics analysis

“…The ilvBN and ilvIH genes are transcribed as separate operons, whereas ilvGM is located within the ilvGMEDA operon. The ilvGMEDA and ilvBN operons are regulated by transcription attenuation, and the leader peptide reading frame of the attenuator contains regulatory codons for all three amino acids, isoleucine, leucine, and valine [9]. The leuABCD operon contains genes for the leucine biosynthesis and expression of this operon also is regulated by transcription attenuation [10].…”