Escherichia coli plasmid vectors containing synthetic translational initiation sequences and ribosome binding sites fused with the lacZ gene

The construction of a series of Escherichia coli plasmid vectors suitable for assaying the effects of gene control signals fused with the E. coli lacZ gene is reported. A synthetic deoxyoligonucleotide dodecamer 5'-CATGAATTCATG GTACTTAAGTAC-5' containing two translation initiation codons (ATG) separated by an EcoRI site was ligated with a lacZ gene derivative which lacks the codons for the first eight amino acids in plasmid pMC1403 (Casadaban et al., 1980). Two ribosome-binding sequences were synthesised and inserted into the EcoRI site before an ATG, and the effects of these sequences on lacZ gene expression in vivo measured by assaying beta-galactosidase activity. The E. coli ribosomal RNA gene (rrnB) promoter, the tetracycline resistance gene promoter, and a lambda phage promoter were cloned using these plasmids. The plasmids are 9.9 kb in size, have ampicillin resistance as a selectable marker and are generally useful for the detection and in vivo assay of gene control regions.     

Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli

Summary The and subunit of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (, and ) or holoenzyme (, , and ⁷⁰) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls synthesis in vivo.     

大肠杆菌E.coli K-12转录因子结合位点的理论预测

基于实验验证的22种大肠杆菌K12的转录因子结合位点序列,分析了转录因子结合位点每一位置的碱基保守性,提出了预测转录因子结合位点的位置权重矩阵打分函数算法(PWMSA)。利用self-consistency和cross-validation两种检验方法对此算法进行检验,self-consistency检验总的预测成功率达到87.59%,cross-validation检验成功率达到85.48%。对基因间序列进行搜索,获得了多个可能的转录因子结合位点。     

The role of the omega subunit of RNA polymerase in expression of the relA gene in Escherichia coli

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level.     

Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc‐binding sites in the C‐terminal domain

Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N‐terminal catalytic domain and a 30 kDa C‐terminal zinc‐binding region (ZD domain) which has three adjacent tetra‐cysteine zinc‐binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zinc‐binding motifs in ZD domain and both the first and second zinc‐binding motifs are required for iron binding in TopA. The site‐directed mutagenesis studies further reveal that while the mutation of the third zinc‐binding motif has very little effect on TopA's activity, mutation of the first two zinc‐binding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo, indicating that the first two zinc‐binding motifs in TopA are crucial for its function. The DNA‐binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single‐stranded (ss) DNA‐binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity.     

Phosphorylation of Escherichia coli poly(A) polymerase I and effects of this modification on the enzyme activity

In Escherichia coli, RNA polyadenylation is catalyzed mainly by poly(A) polymerase I (PAP I). Here we demonstrate that a PAP I variant with a C-terminal His tag (PAP I-His) can be phosphorylated both in vivo and in an artificial in vitro system. The in vivo phosphorylation of PAP I-His impairs activity of this enzyme. Previous studies, performed by others, indicated that phosphorylation of His-tagged proteins usually reflects such a modification of their native counterparts in bacterial cells. Therefore, our results suggest that phosphorylation and dephosphorylation of PAP I may be important regulatory processes in the control of activity of this enzyme.