TrfA dimers play a role in copy-number control of RK2 replication.
TrfA dimers play a role in copy-number control of RK2 replication.
复制标题
TrfA 二聚体在 RK2 复制的拷贝数控制中发挥作用。
DOI：
10.1016/s0378-1119(98)00370-9
复制
发表时间：
1998
期刊：
影响因子：
3.5
通讯作者：
Helinski,DR
中科院分区：
文献类型：
--
作者：
Toukdarian,AE;Helinski,DR
广宿主范围质粒RK 2的拷贝数调节依赖于质粒编码的起始蛋白TrfA和RK 2复制起点。拷贝数控制的手铐模型提出，TrfA结合的oris可逆地耦合，以防止进一步启动质粒复制时，在体内的拷贝数是在复制子特异性拷贝数或以上。已经分离出允许oriV以升高的拷贝数复制的TrfA突变体。为了更好地理解“手铐”的机制，进一步表征了拷贝上调的TrfA（G254 D/S267 L）突变体。在本研究中，我们通过尺寸排阻色谱法和天然凝胶电泳显示，与wt TrfA主要是二聚体不同，纯化的His 6-TrfA（G254 D/S267 L）主要是单体。在体内，TrfA 33（G254 D/S267 L）支持RK 2 ori质粒以大大提高的拷贝数反式复制，而顺式质粒表现出失控复制。然而，在用编码TrfA 33（G254 D/S267 L）的微型RK 2复制子转化的细胞中，先前分离的两种DNA结合缺陷型TrfA突变体TrfA 33（P151 S）或TrfA 33（S257 F）中的任一种的表达导致失控表型的抑制。His 6-TrfA（P151 S）和His 6-TrfA（S257 F）纯化为二聚体，并且当在体内表达时不能支持RK 2质粒复制。相比之下，trfA（P151 S）或trfA（S257 F）突变与trfA（G254 D/S267 L）突变的组合导致突变体TrfA蛋白的表达，所述突变体TrfA蛋白主要是单体并且不再能够在体内恢复对由TrfA 33（G254 D/S267 L）指导的复制的拷贝控制。基于这些发现，提出了一种手铐模型，其中oriV结合的TrfA单体通过二聚体TrfA分子偶联。
Copy-number regulation of the broad-host-range plasmid RK2 is dependent on the plasmid-encoded initiator protein, TrfA, and the RK2 origin of replication. The handcuffing model for copy-number control proposes that TrfA-bound oris reversibly couple to prevent the further initiation of plasmid replication when the copy number in vivo is at or above the replicon-specific copy number. TrfA mutants have been isolated which allow for oriV replication at elevated copy numbers. To better understand the mechanism of `handcuffing', the copy-up TrfA(G254D/S267L) mutant was characterized further. In the present study we show by size exclusion chromatography and native gel electrophoresis that unlike wt TrfA which is largely dimeric, purified His6-TrfA(G254D/S267L) is primarily monomeric. In vivo, TrfA33(G254D/S267L) supports replication of an RK2 ori plasmid in trans at a greatly elevated copy number, while in cis the plasmid exhibits runaway replication. However, expression of either of two previously isolated DNA-binding defective TrfA mutants, TrfA33(P151S) or TrfA33(S257F), in a cell transformed with a mini-RK2 replicon encoding TrfA33(G254D/S267L) results in suppression of the runaway phenotype. His6-TrfA(P151S) and His6-TrfA(S257F) purify as dimers, and when expressed in vivo are incapable of supporting RK2 plasmid replication. In contrast, combination of the trfA(P151S) or trfA(S257F) mutation with the trfA(G254D/S267L) mutations results in the expression of mutant TrfA proteins which are mainly monomers and which can no longer restore copy control to replication directed by TrfA33(G254D/S267L) in vivo. On the basis of these findings a handcuffing model is proposed, whereby oriV-bound TrfA monomers are coupled by dimeric TrfA molecules.