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BkdR is the transcriptional activator of thebkd operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex ofPseudomonas putida . In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5′ region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of thebkd operon was obtained by using a vector containing thebkdR-bkdA1 intergenic region plus the putative ρ-independent terminator of thebkd operon. Substrate DNA, BkdR, and any of thel -branched-chain amino acids ord -leucine was required for transcription. Branched-chain keto acids,d -valine, andd -isoleucine did not promote transcription. Therefore, thel -branched-chain amino acids andd -leucine are the inducers of thebkd operon. The concentration ofl -valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of thebkd operon by BkdR during enzyme induction which incorporates these results is presented.

journal of bacteriology

In Vitro Transcriptional Studies of the <i>bkd</i> Operon of <i>Pseudomonas putida</i> : <scp>l</scp> -Branched-Chain Amino Acids and <scp>d</scp> -Leucine Are the Inducers

In Vitro Transcriptional Studies of the bkd Operon of Pseudomonas putida : l -Branched-Chain Amino Acids and d -Leucine Are the Inducers