Malys Naglis, Chang Dau-Yin, Baumann Richard G, Xie Dongmei, Black Lindsay W
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201-1503, USA.
J Mol Biol. 2002 May 31;319(2):289-304. doi: 10.1016/S0022-2836(02)00298-X.
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 approximately 10% 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.
HOC和SOC是噬菌体T4衣壳的非必需蛋白，可用于噬菌体展示多个拷贝的肽和蛋白质。通过在四丙氨酸连接肽上展示来自SOC羧基末端的五个随机氨基酸和来自HOC氨基末端的五个随机氨基酸，构建了一个二分体噬菌体T4肽库。该二分体文库针对噬菌体T4末端酶大亚基gp17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。所选噬菌体展示的序列与那些先前通过遗传和生化标准已知与gp17相互作用的T4基因产物相匹配：gp20（门户蛋白）、gp32（单链DNA结合蛋白）、gp16（末端酶小亚基）和gp17（自身）。此外，还发现与gp55（T4晚期σ因子）、gp45（滑动夹钳）、gp44（夹钳装载器）、gp2（DNA末端蛋白）和gp23（主要衣壳蛋白）相匹配。在gp55的118 - 134氨基酸区域发现了大量的氨基酸序列匹配。免疫沉淀和亲和柱层析证明了gp17和gp55的直接结合；此外，gp17特异性结合与gp55残基111 - 136相对应的柱偶联肽。对突变体感染细菌中基因17和其他mRNA水平的测量不支持gp17 - gp55相互作用在调节末端酶或其他晚期基因转录中的作用。然而，虽然在前头部和末端酶缺陷的噬菌体T4感染中积累的DNA串联体可以在体外以约10%的野生型效率进行包装，但55am33am缺陷的串联体DNA包装效率至少低100倍。此外，gp55残基111 - 136肽在体外特异性阻断DNA包装。这些结果表明，T4末端酶与T4晚期σ因子gp55的相互作用在体内DNA包装中起作用。gp55相互作用可能起到将末端酶加载到DNA上进行包装的作用。
