Roof W D, Horne S M, Young K D, Young R
Department of Biochemistry and Biophysics, Texas A & M University, College Station 77843.
J Biol Chem. 1994 Jan 28;269(4):2902-10.
Recessive mutations in the slyD gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage phi X174 (Maratea, D., Young, K., and Young, R. (1985) Gene (Amst.) 40, 39-46). The slyD1 mutation, transduced into the normal phi X174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function rather than for expression from the plasmid vector. The cloning, sequencing, and deletion analysis of a 1-kilobase pair genomic fragment containing the slyD locus, mapping at 73.5', is reported. Three reading frames, orf72, orf159, and orf196, are contained within this fragment, with the latter two reading frames occupying the same DNA on opposite strands. Deletion analysis shows that the complementing activity is restricted to the orf159/orf196 DNA. Complementation of the SlyD phenotype was observed irrespective of the orientation of the orf159/orf196 DNA with respect to a vector promoter, indicating that a cryptic promoter serves slyD on this fragment. Using site-directed mutagenesis, nonsense mutations were created in each reading frame which were silent in the opposing frame. Both orf196 nonsense alleles failed to complement slyD1, whereas both orf159 nonsense alleles retained complementation, demonstrating rigorously that orf196 is slyD. A segment corresponding to the first 150 residues of the predicted SlyD protein has significant similarity throughout its length to the FKBP family of peptidyl-prolyl cis-trans-isomerases or rotamases. The COOH-terminal 46 codons of slyD encode a remarkable histidine-rich peptide sequence which is at least partly dispensable for slyD function in E-mediated lysis. Overexpression of slyD in E. coli is toxic. These findings are discussed in terms of a model for SlyD involvement in E function and in terms of a model for SlyD involvement of the ubiquitous FKBP rotamases.
通过在诱导噬菌体φX174的克隆裂解基因E后选择存活来分离slyD基因中的隐性突变（Maratea, D., Young, K., and Young, R. (1985) Gene (Amst.) 40, 39 - 46）。将slyD1突变转导到正常的φX174宿主大肠杆菌C中，对野生型噬菌体的噬菌斑形成能力产生绝对阻断，表明slyD是E功能所必需的，而非质粒载体表达所必需。本文报道了一个包含slyD基因座（定位在73.5'）的1千碱基对基因组片段的克隆、测序及缺失分析。该片段包含三个阅读框，即orf72、orf159和orf196，后两个阅读框位于相反链上的相同DNA区域。缺失分析表明互补活性局限于orf159/orf196 DNA。无论orf159/orf196 DNA相对于载体启动子的方向如何，均观察到SlyD表型的互补，这表明该片段上存在一个隐性启动子为slyD提供服务。利用定点诱变，在每个阅读框中创建了在相反阅读框中沉默的无义突变。两个orf196无义等位基因均不能互补slyD1，而两个orf159无义等位基因保留互补能力，这严格证明了orf196就是slyD。对应于预测的SlyD蛋白前150个残基的一段序列在其全长上与肽基脯氨酰顺反异构酶或旋转异构酶的FKBP家族具有显著相似性。slyD的羧基末端46个密码子编码一个显著富含组氨酸的肽序列，该序列对于slyD在E介导的裂解中的功能至少部分是可有可无的。在大肠杆菌中过表达slyD是有毒的。本文从slyD参与E功能的模型以及slyD参与普遍存在的FKBP旋转异构酶的模型的角度对这些发现进行了讨论。
