The specificity of attenuation control of the expression of theilvGMEDA operon in Escherichia coli K-12

Jiun Wei Chen, Purdue University

Abstract

Three different approaches were used to determine the regulatory effects of amino acids on the expression of ilvGMEDA operon in Escherichia coli. A burst of enzyme synthesis immediately after the relief of amino acid starvation reflected the accumulation of initiated ilv mRNA chains on which translation had been initiated and ribosomes were stalled at codons for the restricted amino acid. Auxotrophic and prototrophic derivatives of an ilv::lac strain were used to determine the response of the ilvGMEDA regulatory region to the supply of amino acids under limiting and repressing conditions respectively. Proline, arginine, and serine, codons for which were interspersed among those for isoleucine, leucine, and valine in the wild type ilvGMEDA leader transcript, were unable to exert any regulatory effect on the expression of ilvGMEDA operon. Neither were any of the amino acids in the ilvGMEDA leader peptide other than the three branched-chain amino acids able to demonstrate significant regulatory effect. A special scheme that utilized a recBC sbcB strain was developed to transfer the mutations generated in vitro efficiently into the ilvGMEDA leader region of the ilv::lac strains. A similar approach was used to replace the entire ilvGMEDA operon with an aad$\sp+$ (Spc$\sp{\rm r}$/Str$\sp{\rm r}$) gene. Six site-specific mutations in the ilvGMEDA leader region were created by synthetic oligonucleotide primed mutagenesis and restriction enzyme digestion. Neither proline, arginine, nor phenylalanine was able to assume the regulatory function of the "regulatory" amino acids which they replaced in the ilvGMEDA leader peptide. Two mutations that have an altered reading frame and a truncated leader peptide with no regulatory amino acids in it, showed high expression of ilvGMEDA operon and loss of multivalent control by isoleucine, leucine, and valine. It was concluded from this study than an attenuation mechanism is the only end-product specific regulatory mechanism affecting the ilvGMEDA operon. Deletion of the terminator region and replacement of the codons for the regulatory amino acids abruptly abolished the regulatory effect of the branched-chain amino acids. No other amino acids studied were able to assume the regulatory effect of the three branched-chain amino acids even when their codons were in the positions normally occupied by the branched-chain amino acids in the leader transcript. Thus the ilv leader region is not only highly specific for responding to the supply of the branched-chain amino acids but also remarkably refractory to changes that would result in regulation by other amino acids.

Degree

Ph.D.

Advisors

Umbarger, Purdue University.

Subject Area

Molecular biology

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