Kurenbach Brigitta, Grothe Dagmar, Farías María Eugenia, Szewzyk Ulrich, Grohmann Elisabeth
Fachgebiet Okologie der Mikroorganismen, Institut für Technischen Umweltschutz, Technische Universität Berlin, D-10587 Berlin, Germany.
J Bacteriol. 2002 Mar;184(6):1801-5. doi: 10.1128/JB.184.6.1801-1805.2002.
The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriT(pIP501). The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg(2+) or Mn(2+) and is highest at temperatures of between 42 and 45C.
通过逆转录聚合酶链反应(RT-PCR)表明,粪肠球菌中pIP501的tra基因orf1至orf11转录为一个11.3 kb的单一操纵子。tra mRNA的转录起始位点定位在操纵子第一个基因traA解旋酶预测的TTG起始密码子上游110 bp处。将TraA蛋白(660个氨基酸)和TraA蛋白的C末端截短版本(293个氨基酸)作为与谷胱甘肽S-转移酶的融合蛋白进行纯化。在含有oriT(pIP501)的超螺旋质粒pVA2241 DNA上,体外证明了两种TraA蛋白的oriT切割活性。DNA解旋酶TraA的活性严格依赖于Mg(2+)或Mn(2+)的存在,并且在42至45℃的温度下活性最高。