Lin H, Rao V B, Black L W
Department of Biochemistry and Molecular Biology, University of Maryland at Baltimore, Baltimore, MD, 21201-1503, USA.
J Mol Biol. 1999 Jun 4;289(2):249-60. doi: 10.1006/jmbi.1999.2781.
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 antigenicoutercapsid 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 interaction sites. In 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.
噬菌体DNA包装是由与前头部的门户顶点十二聚体复合的噬菌体末端酶将串联DNA通过ATP驱动转运到前头部中实现的。通过突变分析和结构基因内的序列定位确定了噬菌体T4末端酶和门户基因20产物（gp20）的功能结构域。通过遗传学（末端酶-门户基因间抑制突变）、生物化学（gp17的柱保留和gp20肽对体外DNA包装的抑制）和免疫学（聚合的gp20肽和gp17的共免疫沉淀）研究确定了门户顶点和大末端酶亚基（gp17）的相互作用区域。通过噬菌体T4 HOC（高抗原性外 capsid 蛋白）展示系统测试了相互作用的特异性，其中野生型、cs20和打乱的门户肽序列展示在噬菌体T4的HOC蛋白上。通过His标签固定的gp17柱对这些重组噬菌体的保留以及与纯化的末端酶的共免疫沉淀确定的这些重组噬菌体的结合亲和力支持了门户蛋白和末端酶相互作用位点的特异性。为了进一步支持特异性，对应于鉴定位点一部分的gp20肽抑制包装，而打乱序列的肽在体外不阻断DNA包装。门户相互作用位点定位在gp20线性序列（524个残基）中央部分的28个残基处。根据两对基因间门户-末端酶抑制突变判断，末端酶大亚基gp17的两个独立区域（中央和COOH末端）通过门户位点的疏水接触相互作用。尽管末端酶显然与这种gp20门户肽相互作用，但针对门户肽的多克隆抗体在天然结构中似乎无法接近它，这表明gp20和gp17的紧密结合可能使末端酶区域在包装体复合物的门户内内化。与在噬菌体T3和λ中鉴定的类似位点相比，既有相似之处也有不同之处。
