Kataoka M, Kosono S, Seki T, Yoshida T
International Center of Cooperative Research in Biotechnology, Faculty of Engineering, Osaka University, Japan.
J Bacteriol. 1994 Dec;176(23):7291-8. doi: 10.1128/jb.176.23.7291-7298.1994.
Expression of the tra operon, essential for conjugative transfer of the 11-kb Streptomyces nigrifaciens plasmid pSN22, is negatively regulated by traR, which is located upstream of the tra operon and transcribed in the opposite orientation. The transcriptional start points for the tra and traR mRNAs were determined by primer extension; they are 72 bp apart and have identical -10 promoter sequences. The TraR protein was overexpressed in Escherichia coli and used for gel retardation and DNase I protection experiments. It bound specifically to the bidirectional tra-traR promoter region and protected four DNA regions, each of which contains a similar 12-bp sequence. The binding was strongest to the region downstream of the tra promoter, probably ensuring that expression of the potentially lethal traB gene is turned off before traR. The efficiency of intramycelial plasmid transfer was decreased by the mutation at the downstream region.
tra操纵子对于11kb的黑色链霉菌质粒pSN22的接合转移至关重要，其表达受到traR的负调控，traR位于tra操纵子上游且转录方向相反。通过引物延伸确定了tra和traR mRNA的转录起始点；它们相距72bp且具有相同的-10启动子序列。TraR蛋白在大肠杆菌中过表达，并用于凝胶阻滞和DNase I保护实验。它特异性结合双向tra-traR启动子区域并保护四个DNA区域，每个区域都包含一个相似的12bp序列。结合在tra启动子下游区域最强，这可能确保潜在致死性的traB基因在traR之前关闭表达。下游区域的突变降低了菌丝内质粒转移的效率。
