Yeh L S, Hsu T, Karam J D
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
J Bacteriol. 1998 Apr;180(8):2005-13. doi: 10.1128/JB.180.8.2005-2013.1998.
The genomes of bacteriophages T4 and RB69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo. We describe here the biological implications of divergence in a genomic segment that encodes four essential DNA replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein). We have cloned, sequenced, and expressed several overlapping segments of the RB69 gene 46-45.2-(rpbA)-45-44-62 cluster and compared its features to those of the homologous gene cluster from T4. The deduced primary structures of all four RB69 replication proteins and gp45.2 from this cluster are very similar (80 to 95% similarity) to those of their respective T4 homologs. In contrast, the rpbA region (which encodes a nonessential protein in T4) is highly diverged (approximately 49% similarity) between the two phage genomes and does not encode protein in RB69. Expression studies and patterns of high divergence of intercistronic nucleotide sequences of this cluster suggest that T4 and RB69 evolved similar transcriptional and translational control strategies for the cistrons contained therein, but with different specificities. In plasmid-phage complementation assays, we show that posttranslationally, RB69 and T4 homologs of gp45 and the gp44/62 complex can be effectively exchanged between the two phage replicase assemblies; however, we also show results which suggest that mixed clamp loader complexes consisting of T4 gp62 and RB69 gp44 subunits are not active for phage DNA replication. Thus, specificity of the gp44-gp62 interaction in the clamp loader marks a point of departure between the T4 and RB69 replication systems.
噬菌体T4和RB69的基因组在系统发育上相关，但在许多位点的核苷酸序列上存在差异，并且在体内彼此不兼容。我们在此描述了一个基因组片段差异的生物学意义，该片段编码四种必需的DNA复制蛋白：gp45（滑动夹钳）、gp44/62复合物（夹钳装载器）和gp46（一种重组蛋白）。我们克隆、测序并表达了RB69基因46 - 45.2 -（rpbA）- 45 - 44 - 62簇的几个重叠片段，并将其特征与T4的同源基因簇进行了比较。该簇中所有四种RB69复制蛋白和gp45.2的推导一级结构与它们各自的T4同源物非常相似（相似性为80%至95%）。相比之下，rpbA区域（在T4中编码一种非必需蛋白）在两个噬菌体基因组之间高度分化（相似性约为49%），并且在RB69中不编码蛋白质。该簇的表达研究和顺反子间核苷酸序列的高度差异模式表明，T4和RB69针对其中所含的顺反子进化出了相似但具有不同特异性的转录和翻译控制策略。在质粒 - 噬菌体互补试验中，我们表明在翻译后，gp45的RB69和T4同源物以及gp44/62复合物可以在两个噬菌体复制酶组装体之间有效交换；然而，我们也展示了一些结果，表明由T4 gp62和RB69 gp44亚基组成的混合夹钳装载器复合物对噬菌体DNA复制无活性。因此夹钳装载器中gp44 - gp62相互作用的特异性标志着T4和RB69复制系统之间的一个分歧点。
