A bipartite bacteriophage T4 SOC and HOC randomized peptide display library:: Detection and analysis of phage T4 terminase (gp17) and late σ factor (gp55) interaction
A bipartite bacteriophage T4 SOC and HOC randomized peptide display library:: Detection and analysis of phage T4 terminase (gp17) and late σ factor (gp55) interaction
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DOI：
10.1016/s0022-2836(02)00298-x
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发表时间：
2002-05-31
影响因子：
5.6
通讯作者：
Black, LW
中科院分区：
文献类型：
--
作者：
Malys, N;Chang, DY;Black, LW
HOC和SOC是可用于肽和蛋白质的多个拷贝的噬菌体展示的CD 4 T4衣壳蛋白。通过在四丙氨酸接头肽上展示来自SOC的羧基末端的五个随机氨基酸和来自HOC的氨基末端的五个随机氨基酸来创建二分噬菌体T4肽文库。对噬菌体T4末端酶大亚基gp 17进行生物淘选，以鉴定可能与末端酶相互作用的T4基因产物。所显示的选定的T4基因产物的序列与先前通过遗传和生物化学标准已知的与gp 17相互作用的那些T4基因产物相匹配：gp 20（门户蛋白）、gp 32（单链DNA结合蛋白）、gp 16（末端酶小亚基）和gp 17（自身）。此外，还发现了与gp 55（T4晚期σ因子）、gp 45（滑动钳）、gp 44（钳加载器）、gp 2（DNA末端蛋白）和gp 23（主要衣壳蛋白）的匹配。免疫沉淀和亲和柱层析证明直接结合的gp 17和gp 55，而且，gp 17结合特异性的柱偶联肽对应于gp 55残基111-136。基因17和其他mRNA水平的测量在muclobulin感染的细菌不支持的作用，gp 17-gp 55的相互作用，调节终止酶或其他晚期基因转录。然而，尽管在前体和末端酶缺陷型噬菌体T4感染中积累的DNA多联体可以在体外包装到类似于10%的野生型效率，但55 am-33 am缺陷型多联体DNA的包装效率至少低100倍。此外，gp 55残基111-136肽在体外特异性地阻断DNA包装。这些结果表明T4末端酶与T4晚西格玛因子gp 55的相互作用在体内DNA包装中起作用。gp 55相互作用可以起到将末端酶装载到DNA上用于包装的作用。(C)2002爱思唯尔科技有限公司。保留所有权利。
HOC and SOC are dispensable T4 capsid proteins that can be used for phage display of multiple copies of peptides and proteins. A bipartite phage T4 peptide library was created by displaying on tetra-alanine linker peptides five randomized amino acids from the carboxyl-terminus of SOC and five randomized amino acids from the amino terminus of HOC. The bipartite library was biopanned against the phage T4 terminase large subunit gp17 to identify T4 gene products that may interact with the terminase. The sequences of selected phages displayed matches to those T4 gene products previously known by genetic and biochemical criteria to interact with gp17: gp20 (portal protein), gp32 (single-stranded DNA binding protein), gp16 (terminase small subunit), and gp17 (self). In addition, matches were found to gp55 (T4 late sigma factor), gp45 (sliding clamp), gp44 (clamp loader), gp2 (DNA end protein), and gp23 (major capsid protein).Abundant amino acid sequence matches were found to aa region 118-134 of gp55. Immunoprecipitation and affinity column chromatography demonstrated direct binding of gp17 and gp55; moreover, gp17 bound specifically to a column-coupled peptide corresponding to gp55 residues 111-136. Measurements of gene 17 and other mRNA levels in mutant-infected bacteria did not support a role of gp17-gp55 interaction in regulation of terminase or other late gene transcription. However, whereas DNA concatemers that accumulate in prohead and terminase defective phage T4 infections could be packaged in vitro to similar to10% wild-type efficiency, 55am33am defective concatemeric DNA was packaged at least 100-fold less efficiently. Moreover, gp55 residues 111-136 peptide specifically blocked DNA packaging in vitro. These results suggest that the T4 terminase interaction with T4 late sigma factor gp55 plays a role in DNA packaging in vivo. The gp55 interaction may function to load the terminase onto DNA for packaging. (C) 2002 Elsevier Science Ltd. All rights reserved.