Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase
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DOI：
10.1073/pnas.1133325100
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发表时间：
2003-08-05
影响因子：
11.1
通讯作者：
Rothman-Denes, LB
中科院分区：
文献类型：
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作者：
Davydova, EK;Rothman-Denes, LB
大肠杆菌噬菌体N4病毒体RNA聚合酶（vRNAP）是RNA聚合酶的T7样家族中最远的成员，负责线性双链DNA噬菌体基因组的早期基因的转录。大肠杆菌单链DNA结合蛋白（EcoSSB）是N4早期体内转录以及在含有vRNAP启动子的超螺旋DNA模板上体外转录所必需的。与其他DNA依赖性RNA聚合酶相比，vRNAP在具有体内特异性的单链、含启动子的模板上启动转录;然而，RNA产物不被置换，因此将模板使用限制为一轮。我们表明，EcoSSB激活vRNAP转录在有限的单链模板浓度通过模板回收。EcoSSB与模板和新生转录物结合，并阻止转录惰性RNA：DNA杂合体的形成。使用C-末端截短的EcoSSB突变体蛋白，人线粒体SSB（Hsmt SSB），噬菌体P1 SSB，和F附加体编码的SSB，以及Hsmt-EcoSSB嵌合体，我们已经映射到EcoSSB的C-末端氨基酸的模板回收的决定因素。T7 RNAP含有一个氨基末端结构域，负责在RNA产物离开酶时结合RNA产物。在vRNAP中不存在与该结构域的序列相似性。因此，我们提出了EcoSSB的独特作用：它在N4 vRNAP中功能上取代了负责RNA结合的T7 RNAP的N-末端结构域。
Coliphage N4 virion RNA polymerase (vRNAP), the most distantly related member of the T7-like family of RNA polymerases, is responsible for transcription of the early genes of the linear double-stranded DNA phage genome. Escherichia coli single-stranded DNA-binding protein (EcoSSB) is required for N4 early transcription in vivo, as well as for in vitro transcription on supercoiled DNA templates containing vRNAP promoters. In contrast to other DNA-dependent RNA polymerases, vRNAP initiates transcription on single-stranded, promoter-containing templates with in vivo specificity; however, the RNA product is not displaced, thus limiting template usage to one round. We show that EcoSSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling. EcoSSB binds to the template and to the nascent transcript and prevents the formation of a transcriptionally inert RNA:DNA hybrid. Using C-terminally truncated EcoSSB mutant proteins, human mitochondrial SSB (Hsmt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hsmt-EcoSSB chimera, we have mapped a determinant of template recycling to the C-terminal amino acids of EcoSSB. T7 RNAP contains an amino-terminal domain responsible for binding the RNA product as it exits from the enzyme. No sequence similarity to this domain exists in vRNAP. Hereby, we propose a unique role for EcoSSB: It functionally substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding.