Analysis of capsid portal protein and terminase functional domains: Interaction sites required for DNA packaging in bacteriophage T4
Analysis of capsid portal protein and terminase functional domains: Interaction sites required for DNA packaging in bacteriophage T4
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DOI：
10.1006/jmbi.1999.2781
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发表时间：
1999-06-04
影响因子：
5.6
通讯作者：
Black, LW
中科院分区：
文献类型：
--
作者：
Lin, HC;Rao, VB;Black, LW
噬菌体DNA包装的结果，从ATP驱动的易位串联DNA到前体的噬菌体末端酶复合的门户顶点十二聚体的前体。通过突变体分析和结构基因内的序列定位确定噬菌体T4末端酶和门户基因20产物（gp 20）的功能结构域。门顶点和大末端酶亚基（gp 17）的相互作用区域通过遗传学（末端酶-门基因间抑制突变）、生物化学（gp 17的柱保留和gp 20肽对体外DNA包装的抑制）和免疫学（聚合gp 20肽和gp 17的免疫共沉淀）研究确定。通过噬菌体T4 HOC（高抗原性外衣壳蛋白）展示系统测试相互作用的特异性，其中野生型、cs20和乱序门户肽序列展示在噬菌体T4的HOC蛋白上。这些重组的结合亲和力，确定通过保留这些结合由His-标签固定的gp 17柱，并通过与纯化的末端酶的免疫共沉淀支持的特定性质的门户蛋白和末端酶相互作用位点。在进一步支持特异性的情况下，对应于鉴定位点的一部分的gp 20肽抑制包装，而乱序肽在体外不阻断DNA包装。门户网站的相互作用位点定位于28个残基的线性序列的gp 20（524个残基）的中心部分。作为判断两对基因间的门户-末端酶抑制突变，两个独立的区域的末端酶大亚基gp 17（中央和COOH-末端）通过疏水性接触在门户网站的相互作用。虽然末端酶显然与此gp 20门户肽相互作用，对门户肽的多克隆抗体似乎无法访问它的天然结构，这表明密切的关联gp 20和gp 17可能内化的门户内的packasome复合物的末端酶区域。相似性和差异都被认为是在比较类似的网站，已确定在λ和λ。(C)北京：科学出版社.
Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenic outer capsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase inter action sites. Ln further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro. The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly internalizes terminase regions within the portal in the packasome complex. Both similarities and differences are seen in comparison to analogous sites which have been identified in phages T3 and lambda. (C) 1999 Academic Press.