Functional dissection of the Bacillus subtilis pur operator site.
Bacillus subtilis PurR represses transcription of several genes involved in purine synthesis, metabolism, and transport and cofactor synthesis. PurR binds specifically to DNAs containing an inverted repeat of a 14-nucleotide "PurBox" located in the upstream control regions of genes in the PurR regulon. Further biochemical investigation of the interaction of PurR with a series of shortened upstream DNA fragments of the pur operon determined the minimum length and specificity elements of the operator. The relative affinities of the two PurBoxes differ significantly, such that upstream PurBox1 (-81 to -68 relative to the transcription start site) is designated "strong" and downstream PurBox2 (-49 to -36) is designated "weak." Two PurBoxes are required for high-affinity PurR binding, and one of these must be strong. The shortest DNA construct with high affinity for PurR is a 74-bp perfect palindrome in which weak PurBox2 and its flanking sequences are replaced by strong PurBox1 and flanking sequences. Two PurR dimers bind to this symmetric construct. Phosphoribosylpyrophosphate (PRPP), the effector molecule that reduces affinity of PurR for DNA, requires one weak PurBox in the DNA construct to inhibit PurR binding. PRPP binds, as expected, to a PRPP-motif in PurR. A tracks outside the central conserved CGAA sequence of the PurBox may facilitate DNA bending, leading to a proposal for strong and weak designations of PurBoxes in the control regions of other genes regulated by PurR.
Bera
Aloke Kumar
AK
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Zhu
Jianghai
J
Zalkin
Howard
H
Smith
Janet L
JL
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D009876:Operon; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D011687:Purines / Q000096:biosynthesis*; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors
D001426:Bacterial Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C065825:PurR protein, Bacteria; D011687:Purines; D012097:Repressor Proteins; D014157:Transcription Factors; D010754:Phosphoribosyl Pyrophosphate