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首页> 外文期刊>Journal of Molecular Biology >Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4
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Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4

机译：来自噬菌体T4的DNA包装/末端酶蛋白gp17的功能分析

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In bacteriophage T4, the terminase complex constituted by the large subunit gp17 (69 kDa) and the small subunit gp16 (18 kDa) is a critical component of the ATP-driven DNA-packaging pump that translocates DNA into an empty capsid shell. Evidence suggests that the large subunit gp17 is the critical component and consists of a number of the functional sites required for DNA-packaging. It exhibits a terminase activity that introduces non-specific cuts into DNA, a portal vertex binding site that allows linkage of cleaved DNA to an empty prohead, an in vitro DNA-packaging activity, and an ATPase activity. In addition, a consensus metal-binding motif and two consensus ATP-binding sites have been identified by sequence analysis. In order to understand the mechanism of action of the multifunctional gp17, we developed an expression-based selection strategy to select for mutants that are defective in terminase function. Characterization of one of the mutants revealed a unique phenotype in which a single H436R mutation resulted in a dramatic loss of both the terminase and the DNA-packaging functions. Indeed, in vivo substitution of H436 with any of the 12 amino acids for which a suppressor is available was lethal to T4 development. According to one hypothesis, H436 is part of a metal-binding motif that is essential for gp17 function. This hypothesis was tested by introducing mutations at each of the three histidine pairs, the H382-X-2-H385 pair, the H411-X-2-H414 pair and the H430X(5)-H436 pair, which constitute the histidine-rich region near the C terminus of gp17. A mutation at either the H411 pair or the H430 pair resulted in a loss of gp17 function, whereas a mutation at the H382 pair had no effect. In addition to the putative metal-binding motif, substitutions at residue K166 within the putative N terminus-proximal Am-binding site also resulted in a loss of gp17 function. We propose that a metal-binding motif involving the histidine residues within the sequence H411-X-2-H414-X-15-H430-X-5-H436 is essential for gp17 function. Metal-terminase interactions may be required for structural alignment and stabilization of functional sites in phage T4 terminase and other double-stranded DNA phage terminases. (C) 1998 Academic Press. [References: 53]
机译：在噬菌体T4中，由大亚基gp17（69 kDa）和小亚基gp16（18 kDa）组成的末端酶复合物是ATP驱动的DNA包装泵的重要组成部分，该泵将DNA转运到空的衣壳中。有证据表明，大的亚基gp17是关键成分，由DNA包装所需的许多功能位点组成。它表现出将非特异性切割引入DNA的末端酶活性，允许裂解的DNA与空前额连接的门户顶点结合位点，体外DNA包装活性和ATPase活性。另外，已经通过序列分析鉴定了共有金属结合基序和两个共有ATP结合位点。为了了解多功能gp17的作用机理，我们开发了一种基于表达的选择策略来选择末端酶功能有缺陷的突变体。突变体之一的表征揭示了独特的表型，其中单个H436R突变导致末端酶和DNA包装功能的急剧丧失。实际上，用抑制子可利用的12种氨基酸中的任何一种在体内替代H436对T4的发展是致命的。根据一个假设，H436是gp17功能必不可少的金属结合基序的一部分。通过在构成组氨酸丰富的三个组氨酸对（H382-X-2-H385对，H411-X-2-H414对和H430X（5）-H436对）中的每一个引入突变来检验该假设gp17 C末端附近的区域。 H411对或H430对的突变导致gp17功能丧失，而H382对的突变没有作用。除了假定的金属结合基序，在假定的N末端近端Am结合位点内的K166残基处的取代也导致gp17功能丧失。我们提出，涉及金属结合基序的序列H411-X-2-H414-X-15-H430-X-5-H436中的组氨酸残基对于gp17功能是必不可少的。金属末端酶相互作用可能是噬菌体T4末端酶和其他双链DNA噬菌体末端酶的结构比对和功能位点稳定所必需的。 （C）1998年学术出版社。 [参考：53]

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