Hurley J M, Chervitz S A, Jarvis T C, Singer B S, Gold L
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309.
J Mol Biol. 1993 Jan 20;229(2):398-418. doi: 10.1006/jmbi.1993.1042.
The acidic carboxy-terminal 89-amino acid fragment of bacteriophage T4 gene 32 protein was expressed in Escherichia coli to high levels from an inducible plasmid construct. Infection of induced cells by wild-type T4 phage results in impaired phage DNA synthesis. The time at which DNA synthesis begins and the diminution in DNA synthesis rates correlate with the amount of carboxy-terminal peptide that accumulates intracellularly prior to infection. Correspondingly, when induced cells are infected with viable phage containing a small deletion near the carboxy-terminus of 32 protein (delta PR201), the inhibition of phage DNA synthesis was much more severe. The mutant 32 protein competes less well against overproduced wild-type acid peptide than does wild-type 32 protein. The purified acid peptide, when used as the attached ligand for affinity chromatography, binds several T4 proteins from phage-infected cells, including 43 protein (T4 DNA polymerase), Dda protein (a DNA helicase), and UvsX protein (a Rec-like recombination protein). Furthermore, at 50- to 100-fold molar excess of acid peptide over intact 32 protein, phage DNA synthesis was specifically inhibited at the initiation step in an in vitro 5-protein DNA replication experiment. We propose that one or more phage replication proteins are titrated as non-productive protein-protein complexes at a site away from the DNA template. This implies that the carboxy-terminal domain of 32 protein is involved in an obligate step of replication machine assembly when the protein is properly attached to ssDNA in the vicinity of a primer-template junction. The assembly defect we observe is strikingly similar to the repression, or "squelching", of the activity of certain eukaryotic transcriptional activators.
噬菌体T4基因32蛋白的酸性羧基末端89个氨基酸片段通过可诱导质粒构建体在大肠杆菌中高水平表达。用野生型T4噬菌体感染诱导细胞会导致噬菌体DNA合成受损。DNA合成开始的时间以及DNA合成速率的降低与感染前细胞内积累的羧基末端肽的量相关。相应地，当用在32蛋白羧基末端附近有小缺失的活噬菌体（δPR201）感染诱导细胞时，噬菌体DNA合成的抑制更为严重。与野生型32蛋白相比，突变型32蛋白与过量产生的野生型酸性肽竞争的能力较差。纯化的酸性肽用作亲和层析的附着配体时，可结合来自噬菌体感染细胞的几种T4蛋白，包括43蛋白（T4 DNA聚合酶）、Dda蛋白（一种DNA解旋酶）和UvsX蛋白（一种Rec样重组蛋白）。此外，在酸性肽相对于完整32蛋白有50至100倍摩尔过量的情况下，在体外5蛋白DNA复制实验的起始步骤中，噬菌体DNA合成受到特异性抑制。我们提出，一种或多种噬菌体复制蛋白在远离DNA模板的位点作为非生产性蛋白-蛋白复合物被滴定。这意味着当32蛋白在引物-模板连接处附近正确附着于单链DNA时，其羧基末端结构域参与复制机器组装的一个必要步骤。我们观察到的组装缺陷与某些真核转录激活因子活性的抑制或“压制”惊人地相似。
