Agrobioteknologiako Instituta, Nafarroako Unibertsitate Publikoa and Consejo Superior de Investigaciones Científicas, Mutiloako etorbidea zenbaki gabe, 31192 Mutiloabeti, Nafarroa, Spain.
Biochem J. 2011 Jan 1;433(1):107-17. doi: 10.1042/BJ20101186.
Although it is generally accepted that Escherichia coli glycogen genes are organized in two tandemly arranged, differentially regulated glgBX and glgCAP operons, RT (reverse transcriptase)-PCR analyses carried out in the present study showed that E. coli cells possess transcripts comprising the five glgBXCAP genes. glg::lacZY expression analyses in cells lacking the region immediately upstream of the glgB gene revealed an almost total abolishment of glgB, glgX and glgC expression, but only a 50-60% reduction of the wild-type glgA and glgP expression levels. Furthermore, similar analyses showed that glgA and glgP expression was almost totally abolished in cells lacking glgA upstream sequences, including glgC, glgB and the asd-glgB intergenic region upstream of glgB. These results indicate that E. coli glgBXCAP genes are organized in a single transcriptional unit controlled by promoter sequences occurring upstream of glgB, and that an alternative suboperonic promoter is located within glgC, driving expression of the glgA and glgP genes. Computer searches for consensus promoters, and analyses of glgB::lacZY and glgA::lacZY expression in cells containing deletions of glgB and glgA upstream sequences identified regions directing glgBXCAP and glgAP expression. 5' RACE (rapid amplification of cDNA ends) analyses located a glgBXCAP transcription start site 155 bp upstream of the glgB initiation codon, and a glgAP transcription start site 359 bp upstream of the glgA initiation codon. Finally, glg::lacZY expression analyses on cells lacking the relA or phoP regulatory genes indicated that both the glgBXCAP operon and the suboperonic promoter driving glgAP expression form part of both the RelA and PhoP-PhoQ regulons.
尽管普遍认为大肠杆菌糖原基因组织在两个串联排列、差异调节的 glgBX 和 glgCAP 操纵子中,但本研究进行的 RT(逆转录)-PCR 分析表明,大肠杆菌细胞具有包含五个 glgBXCAP 基因的转录物。在缺乏 glgB 基因上游区域的细胞中进行的 glg::lacZY 表达分析显示,glgB、glgX 和 glgC 的表达几乎完全被阻断,但 glgA 和 glgP 的表达水平仅降低了 50-60%。此外,类似的分析表明,在缺乏 glgA 上游序列的细胞中,包括 glgC、glgB 和 glgB 上游的 asd-glgB 基因间区,glgA 和 glgP 的表达几乎完全被阻断。这些结果表明,大肠杆菌 glgBXCAP 基因组织在一个由 glgB 上游启动子序列控制的单个转录单元中,并且在 glgC 内存在替代亚操纵子启动子,驱动 glgA 和 glgP 基因的表达。对共识启动子的计算机搜索以及含有 glgB 和 glgA 上游序列缺失的细胞中 glgB::lacZY 和 glgA::lacZY 表达的分析确定了指导 glgBXCAP 和 glgAP 表达的区域。5' RACE(快速扩增 cDNA 末端)分析将 glgBXCAP 转录起始位点定位在 glgB 起始密码子上游 155 bp 处,glgAP 转录起始位点定位在 glgA 起始密码子上游 359 bp 处。最后,在缺乏 relA 或 phoP 调节基因的细胞中进行的 glg::lacZY 表达分析表明,glgBXCAP 操纵子和驱动 glgAP 表达的亚操纵子启动子都构成了 RelA 和 PhoP-PhoQ 调控子的一部分。