d(GC)(n) repeats form Z-DNA within promoter region and repress the promoter activity in Escherichia coli
d(GC)(n) repeats form Z-DNA within promoter region and repress the promoter activity in Escherichia coli
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d(GC)(n) 在启动子区域内重复形成 Z-DNA 并抑制大肠杆菌中的启动子活性
DOI：
10.1093/abbs/gmv038
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发表时间：
2015
影响因子：
3.7
通讯作者：
Hu Chengyu
中科院分区：
文献类型：
--
作者：
Huang Shenghe;Wu Chuxin;Li Dongming;Wang Haizhou;Rao Zechang;Shen Qiling;Chen Chunxiang;Liu Yong;Xu Xun;Hu Chengyu
Z-DNA is a left-handed helical form of DNA in which sugarphosphate backbone winds in a zig–zag pattern. DNA containing alternating purine and pyrimidine repeats has the potential to adopt the Z-DNA formation. In vivo, Z-DNA is formed and stabilized by negative supercoil or specific Z-DNA-binding proteins. Z-DNA-forming sequences are widespread in cells, especially in the promoter regions and centromere of chromatins, so it is thought to be involved in some biological processes such as gene expression, genetic instability, and DNA processing events [1]. Evidence about the effects of Z-DNA on gene transcription regulation is accumulating. Many studies showed that Z-DNA is closely related to gene transcription. On one hand, gene transcription can induce Z-DNA conformation because this process results in negative supercoiling behind a moving RNA polymerase, and Z-DNA form is facilitated at permissive regions. Three fragments of c-MYC promoter were found to bind to Z-DNA antibody only when the gene was actively transcribed [2]. On the other hand, Z-DNA in promoter can regulate gene transcription activity. Rat nucleolin (Ncl) promoter activity is increased by 36%–54% when Z1 [(CA) 10 (CG) 8](at− 631 site) was deleted [3]. However, Z-DNA-forming sequence (at− 815 site) increases the transcription level of Escherichia coli gyrA gene [4]. Therefore, the mechanism how Z-DNA sequence within promoter region modulates gene transcription is complicated, and the effect may be associated with the distance between Z-DNA sequence and certain cis-acting element. The extent of up-regulation is determined by its separation distance relative to TATA box in CYC1 promoter in yeast [5]. It is possible that Z-DNA might regulate gene transcription by various approaches. To test whether Z-DNA-forming sequences could play a regulatory role in gene transcription, a 125bp fragment from mreB (β-actin) promoter P3 (constitutive promoter) was cloned and plasmid pET-Pm-luc was constructed (Supplementary Fig. S1). OverlappingPCR was performed for displacing relevant oligonucleotides with d (GC) 5 or d (GC) 10 at− 70,− 45,− 20, and+ 15 sites from the transcription start site (TSS)(Supplementary Fig. S2) of mreB promoter P3. PCR product was subcloned into BglII/SalI sites of pET-Pm-luc vector to replace mreB promoter P3. The primers used in this study are listed in Supplementary Table S1. Vectors were transformed into E. coli BL21 cells using heat shock method as described in Molecular Cloning (3rd edition, 2001). Cultures of the transformed E. coli cells were diluted from∼ 0.7 to 0.5 of OD600. Luciferase activity was measured on a Hitachi fluorescence spectrophotometer. When the number of repeats in d (GC) n was increased, the inhibitory effect was more obvious (Fig. 1 A); the inhibitory effect of d (GC) 5 replaced at− 45 and+ 15 sites of mreB promoter was much weaker than that of d (GC) 10 (Fig. 1A). Furthermore, the effects of the identical d (GC) n substituted at different sites of promoters on gene transcription were different. For instance, other than that at− 45 and+ 15 sites, d (GC) 5 or d (GC) 10 substituted at− 70 and− 20 sites was inefficient on the promoter activity of mreB (Fig. 1A). These results suggested that effects of d (GC) n on promoter activity were correlated with the length of d (GC) n and substitution sites.
