Upstream CRP-binding site is not essential for CRP-cAMP-mediated inhibition on the nifU promoter  

作　　者：LI Zhiting ZHANG Weijia WANG Yiping 

机构地区：[1]National Laboratory of Protein Engineering and Plant Genetic Engineering,College of Life Sciences,Peking University,Beijing 100871,China

出　　处：《Chinese Science Bulletin》2002年第21期1821-1825,共5页

基　　金：This work was supported in part by the National Natural Science Foundation of China (Grant No. 39925017), the International Cooperation Program (Grant No. 2001AA214211), the "863" Program (Grant No. 2001AA214021) and the "973" Program (Grant No. 2001C

摘　　要：When the NifA-mediated activation of Kleb-siella pneumontae nifU promoter is recreated in Escherichia coli, it has been observed that CRP-cAMP has an inhibitory effect on the nifU promoter. Sequence analysis indicates that there is a strong CRP-binding site located upstream of the nifU promoter, overlapping completely with a previously identified Nif A-binding site. In vitro gel retardation analysis indicates that this putative CRP-binding site has similar affinity for CRP, when compared with that at the lac promoter, suggesting that CRP could effectively compete with NifA for such a binding site under physiological conditions. When this putative CRP-binding site on nifU was mutated, in vitro gel retardation analysis indicates that CRP can no longer bind to the mutant promoter. However, when constitutively expressed NifA is used as the activator, CRP-cAMP-mediated inhibitory effect on this mutant nifU promoter has no significant difference when compared with that obtained from its wild-type promoter.When the NifA-mediated activation of Klebsiella pneumoniae nifU promoter is recreated in Escherichia coli, it has been observed that CRP-cAMP has an inhibitory effect on the nifU promoter. Sequence analysis indicates that there is a strong CRP-binding site located upstream of the nifU promoter, overlapping completely with a previously identified NifA-binding site. In vitro gel retardation analysis indicates that this putative CRP-binding site has similar affinity for CRP, when compared with that at the lac promoter, suggesting that CRP could effectively compete with NifA for such a binding site under physiological conditions. When this putative CRP-binding site on nifU was mutated, in vitro gel retardation analysis indicates that CRP can no longer bind to the mutant promoter. However, when constitutively expressed NifA is used as the activator, CRP-cAMP-mediated inhibitory effect on this mutant nifU promoter has no significant difference when compared with that obtained from its wild-type promoter. These results suggest that direct interaction between CRP and Eσ54, other than the DNA binding site(s) competition between CRP and NifA, plays the principal role in the CRP-cAMP-mediated inhibitory effect on nifU.

关 键 词：CRP-cAMP the nifU PROMOTER gene regulation. 

分 类 号：Q753[生物学—分子生物学]