In vitro transcriptional studies of the bkd operon of Pseudomonas putida: L-branched-chain amino acids and D-leucine are the inducers.
BkdR is the transcriptional activator of the bkd operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of Pseudomonas 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 the bkd operon was obtained by using a vector containing the bkdR-bkdA1 intergenic region plus the putative rho-independent terminator of the bkd operon. Substrate DNA, BkdR, and any of the L-branched-chain amino acids or D-leucine was required for transcription. Branched-chain keto acids, D-valine, and D-isoleucine did not promote transcription. Therefore, the L-branched-chain amino acids and D-leucine are the inducers of the bkd operon. The concentration of L-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 the bkd operon by BkdR during enzyme induction which incorporates these results is presented.
Madhusudhan
K T
KT
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Luo
J
J
Sokatch
J R
JR
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002474:Cell-Free System; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D007658:Ketone Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D007930:Leucine / Q000494:pharmacology*; D051756:Leucine-Responsive Regulatory Protein; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D009876:Operon; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D013237:Stereoisomerism; D014157:Transcription Factors; D014158:Transcription, Genetic
D000597:Amino Acids, Branched-Chain; D004268:DNA-Binding Proteins; D009097:Multienzyme Complexes; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D007930:Leucine