Rao V B, Mitchell M S
Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
J Mol Biol. 2001 Nov 30;314(3):401-11. doi: 10.1006/jmbi.2001.5169.
Double-stranded DNA packaging in bacteriophages is apparently driven by the most powerful molecular motor ever measured. Although it is widely accepted that a translocating ATPase powers the DNA packaging machine, the identity of the ATPase that generates this driving force is unknown. Evidence suggests that the large terminase protein gp17, which possesses two consensus ATP binding motifs and an ATPase activity, is a strong candidate for the translocating ATPase in bacteriophage T4. This hypothesis was tested by a PCR-directed combinatorial mutagenesis approach in which mutant libraries consisting of all possible codon combinations were constructed at the signature residues of the ATP binding motifs. The impact on gp17 function of each randomly selected mutant was evaluated by phenotypic analysis following recombinational transfer into the viral genome. The precise mutation giving rise to a particular phenotype was determined by DNA sequencing. The data showed that the N-terminal ATP binding site I (SRQLGKT(161-167)), but not the ATP binding site II (TAAVEGKS(299-306)), is critical for gp17 function. Even conservative substitutions such as G165A, K166R, and T167A were not tolerated at the GKT signature residues, which are predicted to interact with the ATP substrate. Biochemical analyses of the mutants showed a complete loss of in vitro DNA packaging activity but not the terminase (DNA-cutting) activity. The purified K166G mutant showed a loss of gp17-ATPase activity. The data, for the first time, implicated a specific ATPase center in the viral dsDNA packaging.
噬菌体中的双链DNA包装显然是由迄今所测最强大的分子马达驱动的。尽管人们普遍认为一种易位ATP酶为DNA包装机器提供动力，但产生这种驱动力的ATP酶的身份尚不清楚。有证据表明，具有两个共有ATP结合基序和ATP酶活性的大末端酶蛋白gp17是噬菌体T4中易位ATP酶的有力候选者。通过PCR定向组合诱变方法对这一假说进行了检验，其中在ATP结合基序的特征性残基处构建了由所有可能密码子组合组成的突变体文库。在重组转移到病毒基因组后，通过表型分析评估每个随机选择的突变体对gp17功能的影响。通过DNA测序确定导致特定表型的精确突变。数据表明，N端ATP结合位点I（SRQLGKT(161 - 167)）而非ATP结合位点II（TAAVEGKS(299 - 306)）对gp17功能至关重要。即使是保守取代，如GKT特征性残基处的G165A、K166R和T167A也不能被耐受，这些残基预计与ATP底物相互作用。对突变体的生化分析表明，体外DNA包装活性完全丧失，但末端酶（DNA切割）活性未丧失。纯化的K166G突变体显示gp17 - ATP酶活性丧失。这些数据首次表明病毒双链DNA包装中存在一个特定的ATP酶中心。
