Transcription of the Bacillus subtilis pur operon is regulated by a purine repressor (PurR)-DNA control site interaction. The pur operon control site has two PurBoxes that are re- quired for high-affinity PurR binding...Transcription of the Bacillus subtilis pur operon is regulated by a purine repressor (PurR)-DNA control site interaction. The pur operon control site has two PurBoxes that are re- quired for high-affinity PurR binding. An upstream, strong-binding PurBox1 is at position –81 to –68 relative to the transcription start site and a downstream weak-binding PurBox2 is at position –49 to –36. We constructed three PurBox1 mutations and the effects on binding of PurR to the control region in vitro and on regulation of pur operon expression in vivo were investigated. The mutations significantly reduced the binding of PurR to control region DNA. In strains with G-75A, G-75T and a five bp deletion (?5) pur operon repression was defective in vivo. In addition in vivo PurR titration was used to confirm that sequences flanking PurBox1 and PurBox2 are re- quired for PurR binding to the pur operon control site.展开更多
The amber mutation sites of 6 purR(am) mutants were determined by cloning and DMA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T)...The amber mutation sites of 6 purR(am) mutants were determined by cloning and DMA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T) and C1155(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.展开更多
基金This work was supported by the National Natural Science Foundation of China(Grant No.30070419).
文摘Transcription of the Bacillus subtilis pur operon is regulated by a purine repressor (PurR)-DNA control site interaction. The pur operon control site has two PurBoxes that are re- quired for high-affinity PurR binding. An upstream, strong-binding PurBox1 is at position –81 to –68 relative to the transcription start site and a downstream weak-binding PurBox2 is at position –49 to –36. We constructed three PurBox1 mutations and the effects on binding of PurR to the control region in vitro and on regulation of pur operon expression in vivo were investigated. The mutations significantly reduced the binding of PurR to control region DNA. In strains with G-75A, G-75T and a five bp deletion (?5) pur operon repression was defective in vivo. In addition in vivo PurR titration was used to confirm that sequences flanking PurBox1 and PurBox2 are re- quired for PurR binding to the pur operon control site.
基金the National Natural Science Foundation of China (Grant No. 39770011).
文摘The amber mutation sites of 6 purR(am) mutants were determined by cloning and DMA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T) and C1155(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.
