Williams Sarah L, Schildbach Joel F
Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Nucleic Acids Res. 2006 Jan 17;34(2):426-35. doi: 10.1093/nar/gkj444. Print 2006.
Prior to conjugative transfer of plasmids, one plasmid strand is cleaved in a site- and strand-specific manner by an enzyme called a relaxase or nickase. In F and related plasmids, an inverted repeat is located near the plasmid strand cleavage site, and others have proposed that the ability of this sequence to form a hairpin when in single-stranded form is important for transfer. Substitutions were introduced into a cloned F oriT region and their effects on plasmid transfer were assessed. For those substitutions that substantially reduced transfer, the results generally correlated with effects on in vitro binding of oligonucleotides to the F TraI relaxase domain rather than with predicted effects on hairpin formation. One substitution shown previously to dramatically reduce both plasmid transfer and in vitro binding to a 17-base oligonucleotide had little apparent effect on binding to a 30-base oligonucleotide that contained the hairpin region. Results from subsequent experiments strongly suggest that the relaxase domain can bind to hairpin oligonucleotides in two distinct manners with different sequence specificities, and that the protein binds the oligonucleotides at the same or overlapping sites.
在质粒进行接合转移之前，一条质粒链会被一种叫做松弛酶或切口酶的酶以位点和链特异性的方式切割。在F质粒及相关质粒中，一个反向重复序列位于质粒链切割位点附近，其他人提出该序列在单链形式时形成发夹结构的能力对转移很重要。将取代引入克隆的F oriT区域，并评估它们对质粒转移的影响。对于那些大幅降低转移的取代，结果通常与对寡核苷酸与F TraI松弛酶结构域的体外结合的影响相关，而不是与对发夹形成的预测影响相关。先前显示能显著降低质粒转移和与17碱基寡核苷酸的体外结合的一个取代，对与包含发夹区域的30碱基寡核苷酸的结合几乎没有明显影响。后续实验结果强烈表明，松弛酶结构域可以以两种具有不同序列特异性的不同方式与发夹寡核苷酸结合，并且该蛋白在相同或重叠位点结合寡核苷酸。
