Murialdo H, Tzamtzis D, Berrú M, Fife W L, Becker A
Department of Medical Genetics and Microbiology, University of Toronto, Ontario, Canada.
Mol Microbiol. 1997 Jun;24(5):937-52. doi: 10.1046/j.1365-2958.1997.4011769.x.
DNA maturation in bacteriophage lambda is the process by which the concatemeric precursor DNA is cleaved at sites called cos to generate mature lambda DNA molecules. These DNA molecules are then packaged into procapsids, the empty capsid precursors. The enzyme that catalyses these events is lambda DNA terminase. It is composed of two subunits, made of 181 and 641 amino acids, the products of genes Nu1 and A, respectively. The product of the FI gene (gpFI) stimulates the formation of an intermediate in capsid assembly called complex II, which contains a procapsid, terminase and DNA. The mechanism of stimulation remains unknown. It has been suggested that gpFI may also stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. Mutants in FI fail to mature and package DNA but, in comparison with other capsid gene mutants, FI mutants are leaky. Second site mutants of FI phages, called 'fin' (for FI independence), bypass the necessity for gpFI. These mutants were originally localized to the region of Nu1 and A and are of two classes: finA includes those that induce the synthesis of fourfold more gene A product (gpA) than wild-type phages, and finB includes those that produce normal amounts of gpA. Whereas all finA mutants analysed map to Nu1, finB mutants have been found both in E and in Nu1. The existence of E mutants able to bypass the necessity for gpFI in vivo shows that gpE and gpFI interact, directly or indirectly. Here we have analysed and sequenced two finA mutants and one finB mutant. All of these map in Nu1. Of the two finA mutants, one corresponds to an Ala163Ser change and the other is a silent mutation. It is likely that the finA mutations alter mRNA conformation in a manner that results in an increase in the efficiency of A mRNA translation. The fourfold increase in gpA synthesis translates into a 10-fold increase in terminase activity. These results show that terminase overproduction is sufficient to bypass the necessity for gpFI and that such an overproduction can be achieved by changes in the efficiency of translation of A due to subtle changes in the sequence upstream of the gene. The finBcs103 mutation is a His-87-->Tyr change in Nu1. Therefore, an alternative way in which to bypass the requirement for gpFI involves an alteration in the structure of gpNu1. It is likely that the altered gpNu1 would increase cleavage and packaging efficiency directly or indirectly. We have determined that DNA cleavage in vivo does not occur in the absence of gpFI. Therefore it seems that gpFI somehow facilitates an otherwise latent capacity of terminase to autoactivate its nucleolytic activity.
噬菌体λ中的DNA成熟是一个过程，通过该过程，串联的前体DNA在称为粘性末端位点（cos）的位置被切割，以产生成熟的λDNA分子。然后这些DNA分子被包装到原衣壳中，原衣壳是衣壳的空前体。催化这些事件的酶是λDNA末端酶。它由两个亚基组成，分别由181个和641个氨基酸组成，分别是基因Nu1和A的产物。FI基因的产物（gpFI）刺激衣壳组装过程中一种称为复合物II的中间体的形成，复合物II包含一个原衣壳、末端酶和DNA。刺激机制仍然未知。有人提出，在没有原衣壳的情况下，gpFI也可能通过增加酶的周转来刺激末端酶介导的cos切割。FI基因的突变体无法成熟和包装DNA，但与其他衣壳基因突变体相比，FI突变体是渗漏的。FI噬菌体的第二位点突变体，称为“fin”（代表FI独立性），绕过了对gpFI的需求。这些突变体最初定位在Nu1和A区域，分为两类：finA包括那些诱导合成的基因A产物（gpA）比野生型噬菌体多四倍的突变体，finB包括那些产生正常量gpA的突变体。虽然分析的所有finA突变体都定位在Nu1，但在E和Nu1中都发现了finB突变体。能够在体内绕过对gpFI需求的E突变体的存在表明gpE和gpFI直接或间接相互作用。在这里，我们分析并测序了两个finA突变体和一个finB突变体。所有这些都定位在Nu1中。在这两个finA突变体中，一个对应于Ala163Ser的变化，另一个是沉默突变。finA突变可能以某种方式改变mRNA构象，从而导致A mRNA翻译效率的提高。gpA合成增加四倍转化为末端酶活性增加十倍。这些结果表明，末端酶的过量产生足以绕过对gpFI的需求，并且这种过量产生可以通过基因上游序列的细微变化导致A翻译效率的改变来实现。finBcs103突变是Nu1中His-87→Tyr的变化。因此，绕过对gpFI需求的另一种方式涉及gpNu1结构的改变。改变后的gpNu1可能直接或间接提高切割和包装效率。我们已经确定，在没有gpFI的情况下，体内不会发生DNA切割。因此，似乎gpFI以某种方式促进了末端酶潜在的自我激活其核酸酶活性的能力。
