Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded by purG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu-tamine and ATP f...Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded by purG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu-tamine and ATP for the de novo purine nucleotide biosynthesis. purG gene is negatively regulated by a repressor-oper-ator system. The O+ purG and OC purG were cloned respectively in vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O + ) and pLBG-2 (OC) were carried out. The hybrid plasmids pLB1933 (O+ ) and pLB1927 (OC) containing 5 control region of purG were constructed and the DNA sequences were determined respectively. DNA se-quences data showed that Oc mutation of purG occurred at the 3rd position of 16 bp PUR box in the 5' control region ( G→A). Gel retardation experiment indicated that the repressor bound well with O+ PUR box, but not with Oc PUR box. The result strongly supported the idea that PUR box is the binding region of represser protein and the 3rd posi-tion base G of PUR box is essential for the binding function with repressor protein.展开更多
Salmonella typhimurium purB encodes adeny-losuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of inosine 5’-monophos-phate (IMP) and also the final reaction in the two-step ...Salmonella typhimurium purB encodes adeny-losuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of inosine 5’-monophos-phate (IMP) and also the final reaction in the two-step sequence from IMP to adenosine monophosphate (AMP). The nucleotide sequence ofpurB was obtained by the genetic map and sequence homologous analysis. The conserved pur operator in purB was identified to be located 185 bp downstream of the initiation codon and overlaps codons 62 - 67 in the protein-coding region. The binding of PurR to this operator was demonstrated by gel retardation experiment and site-directed mutagenesis, indicating that the purB is under the control of purR. We also answered why previous study had conflicting report concerning the regulation of purB by purR by identifying the junction site of purB to lacZ in a purB-MudJ (lacZ, Kan’) fusion strain. This result strongly supports that the purB is the second gene in the ycfC-purB operon.展开更多
基金Project supported by the National Natural Science Foundation of China.
文摘Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded by purG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu-tamine and ATP for the de novo purine nucleotide biosynthesis. purG gene is negatively regulated by a repressor-oper-ator system. The O+ purG and OC purG were cloned respectively in vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O + ) and pLBG-2 (OC) were carried out. The hybrid plasmids pLB1933 (O+ ) and pLB1927 (OC) containing 5 control region of purG were constructed and the DNA sequences were determined respectively. DNA se-quences data showed that Oc mutation of purG occurred at the 3rd position of 16 bp PUR box in the 5' control region ( G→A). Gel retardation experiment indicated that the repressor bound well with O+ PUR box, but not with Oc PUR box. The result strongly supported the idea that PUR box is the binding region of represser protein and the 3rd posi-tion base G of PUR box is essential for the binding function with repressor protein.
基金Thiswork was supported by the National Natural Science Foundation of China.
文摘Salmonella typhimurium purB encodes adeny-losuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of inosine 5’-monophos-phate (IMP) and also the final reaction in the two-step sequence from IMP to adenosine monophosphate (AMP). The nucleotide sequence ofpurB was obtained by the genetic map and sequence homologous analysis. The conserved pur operator in purB was identified to be located 185 bp downstream of the initiation codon and overlaps codons 62 - 67 in the protein-coding region. The binding of PurR to this operator was demonstrated by gel retardation experiment and site-directed mutagenesis, indicating that the purB is under the control of purR. We also answered why previous study had conflicting report concerning the regulation of purB by purR by identifying the junction site of purB to lacZ in a purB-MudJ (lacZ, Kan’) fusion strain. This result strongly supports that the purB is the second gene in the ycfC-purB operon.
