| Summary: | 碩士 === 臺灣大學 === 農業化學研究所 === 96 === This study was investigated the regulation of grxD gene expression in Escherichia coli. Based on different lengths of promoter fragment in grxD-lacZ operon and protein fusion, we have found that the gene expression was increased upon entry into stationary phase, and the expression of grxD was enhanced by two promoters P1grxD and P2grxD. While either promoter mutated at the location of -35 (P1grxD) and -10 (P2grxD) respectively, the promoter activity was decreased. In log phase, the grxD was more activated with P1grxD promoter than that with P2grxD.
Under anaerobic condition, the grxD-lacZ gene expression is reduced with both promoters P1grxD and P2grxD. However, it is up-regulated with the one carrying a deletion of unknown conserved region (UCR). In fnr- strain, the expression of grxD-lacZ gene is increased and we suggest that the grxD gene expression is directly repressed by FNR. These findings implied that more than one FNR cis-acting sites are present in the promoter region of grxD.
In fur-, ryhB- or double mutant, we found that the grxD-lacZ gene expression is reduced by RyhB, but Fur still up-regulates the expression even upon iron depletion. When deleted the UCR region, RyhB do not affect the expression level even under iron depletion or LB condtion. We suggest that the UCR region is a RyhB binding site.
Furthermore, we use yeast two-hybrid method to reveal the Grx4 function in vivo. We confirmed that Grx4 interact with Grx1 and TrxB in vivo, and also found Grx4 interact strongly with itself and BolA, and slightly interact with Grx2 and TrxA.
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