Kuebler D, Rao V B
Department of Biology, The Catholic University of America, Washington, DC, 20064, USA.
J Mol Biol. 1998 Sep 4;281(5):803-14. doi: 10.1006/jmbi.1998.1952.
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-X2-H385 pair, the H411-X2-H414 pair and the H430-X5-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 ATP-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-X2-H414-X15-H430-X5-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.
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包装泵的关键组成部分，该泵将DNA转运到空的衣壳壳中。有证据表明，大亚基gp17是关键组成部分，由DNA包装所需的多个功能位点组成。它具有将非特异性切口引入DNA的末端酶活性、允许切割的DNA与空的原头部连接的门户顶点结合位点、体外DNA包装活性和ATP酶活性。此外，通过序列分析鉴定出一个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的作用机制，我们开发了一种基于表达的筛选策略，以筛选末端酶功能有缺陷的突变体。对其中一个突变体的表征揭示了一种独特的表型，其中单个H436R突变导致末端酶和DNA包装功能的显著丧失。事实上，在体内将H436替换为任何一种有抑制子可用的12种氨基酸中的任何一种对T4发育都是致命的。根据一种假设，H436是gp17功能所必需的金属结合基序的一部分。通过在构成gp17 C末端附近富含组氨酸区域的三对组氨酸中的每一对引入突变来检验这一假设，这三对组氨酸分别是H382-X2-H385对、H411-X2-H414对和H430-X5-H436对。H411对或H430对中的任何一个突变都会导致gp17功能丧失，而H382对中的突变则没有影响。除了假定的金属结合基序外，假定的N末端近端ATP结合位点内的K166残基替换也导致gp17功能丧失。我们提出，涉及序列H411-X2-H414-X15-H430-X5-H436内组氨酸残基的金属结合基序对gp17功能至关重要。金属与末端酶的相互作用可能是噬菌体T4末端酶和其他双链DNA噬菌体末端酶功能位点的结构排列和稳定所必需的。
