Biotinyl 5'-adenylate: corepressor role in the regulation of the biotin genes of Escherichia coli K-12.

Abstract

A DNA filter-binding technique was used to study the interaction of the biotin repressor and operator site. From a biotin saturation curve, the concentration for halfmaximal binding (Ko.5) was calculated to be 1 MM. However, in a similar study with the in vitro coupled transcription-translation system in which biotin served as the corepressor, the K0.5 for repression was 7.1 nM. This marked difference of over 2 orders of magnitude was attributed to the activation of biotin by the partially purified repressor preparation in the in vitro system. The activated product formed from biotin, ATP, and repressor preparation was identified as biotinyl 5'-adenylate by paper chromatography and hydroxamic acid formation. Synthetic biotinyl 5'-adenylate was as effective as biotin in the in vitro system (Ko.s, 10 nM) and much more effective than biotin in the DNA-binding assay (Ko.5 1.1 nM versus 1 M&M). These studies indicate that biotinyl 5'-adenylate has a more direct role in the regulation of the biotin genes than does biotin per se.A cluster of five genes coding for the enzymes of the biotin biosynthetic pathway is located at min 17 on the chromosomal map of Escherichia coli (1). These genes are transcribed divergently (2) and their expression is coordinately regulated by biotin through repression (3). The repressive action of biotin is mediated through the bioR gene situated at min 89 close to the bfe locus (4, 5).We have previously described an in vitro coupled transcription-translation system for synthesis of two of the enzymes of the biotin gene cluster; 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase, encoded by A and D genes on the I and r strands, respectively (6). Biotin was effective in repressing the synthesis of both enzymes only in the presence of a partially purified repressor preparation. The specificity for biotin was demonstrated by the inability of various biotin analogs, both natural and synthetic, to function as corepressors. In a preliminary study of the direct interaction of the biotin-repressor complex with the operator site on the DNA by using the DNA filter-binding technique (7, Preparation of DNA. 32P-Labeled X bNo DNA was prepared from a heat-inducible phage, Xc1857bioABFCDs7, kindly supplied by Max Gottesman. A biotin deletion mutant of E. coli K-12, T5-2, was grown to a density of 4 X 108 cells per ml in a low-phosphate medium (pH 7.3) containing per liter: 10 g of neopeptone, 2.5 g of yeast extract, 5 g of NaCl, 2 g of glucose, 5 ,ug of biotin, 5 mg of thiamine, and 1.2 g of MgSO4. The phage lysate was then added to give a multiplicity of infection of 3 and the incubation was continued until a cell density of 1.2 X 109 per ml was attained. After the addition of 10 mCi of [32P]orthophosphoric acid, the temperature of the culture was shifted to 42°C for 15 min for phage induction and then returned to 34°C for an additional 3-hr incubation.*The cells were collected by centrifugation, resuspended in 40 ml of phage buffer (10 mM Tris-HCl, pH 7.3/2 mM MgSO4/68 mM NaCl...

Nucleation of an Allosteric Response via Ligand-induced Loop Folding

“…The repressor binds substrates biotin and ATP to catalyze synthesis of biotinyl-5'-adenylate or bio-5'-AMP(10). The adenylate-bound or activated holomonomer self-associates and the resulting dimer then binds to the biotin operator sequence, bioO, to repress transcription initiation at the biotin biosynthetic operon (11,13). Bio-5'-AMP binding initiates the allosteric signal that is propagated through the BirA monomer to enhance dimerization.…”

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis

“…Transcription of the bio genes is controlled from this bidirectional promoteroperator sequence, and the detection of additional, internal promoters has not been reported. This regulatory model has been confirmed by several methods, including isolation of mutations in the operator region or the birA gene that deregulated biotin production (4,5,9,15,17,21), DNA binding studies of purified BirA to the E. coli bio operon, and DNA protection (5,11,23).…”

“…Transcription of the bio genes is controlled from this bidirectional promoteroperator sequence, and the detection of additional, internal promoters has not been reported. This regulatory model has been confirmed by several methods, including isolation of mutations in the operator region or the birA gene that deregulated biotin production (4,5,9,15,17,21), DNA binding studies of purified BirA to the E. coli bio operon, and DNA protection (5,11,23).In gram-positive bacteria, biotin synthesis has been studied extensively in two species, Bacillus sphaericus (14,16,22) and, more recently, Bacillus subtilis (8,6,7). In B. sphaericus, the genes are located in two separate operons, bioXWF and bioDAYB.…”

Allosteric Signaling in the Biotin Repressor Occurs via Local Folding Coupled to Global Dampening of Protein Dynamics

“…The active BirA species in both functions is bound to bio-5'-AMP, which is synthesized from substrates biotin and ATP (8,9). The adenylated biotin serves both as an intermediate in the biotin transfer reaction and as a corepressor in assembly of the BirA .…”