Adams G M, Blumenthal R M
Department of Microbiology, Medical College of Ohio, Toledo 43699-0008, USA.
Gene. 1995 May 19;157(1-2):193-9. doi: 10.1016/0378-1119(94)00704-v.
The PvuII restriction-modification system has been found to contain three genes which code for a DNA methyltransferase (MTase), a restriction endonuclease (ENase) and a small protein required for expression of the ENase-encoding gene. In addition, there is a small open reading frame (ORF) within and opposite to the MTase-encoding gene. The region containing this ORF is transcribed, and the ORF has an excellent Shine-Dalgarno sequence with an ATA start codon. A closely related ORF is present in the SmaI system. The 28-amino-acid (aa) predicted peptide from the PvuII ORF resembles a region of the PvuII ENase at the dimer interface. We have cloned this ORF, giving it an ATG start codon and putting it under the control of an inducible promoter: induction leads to a slight but significant decrease in restriction of bacteriophage lambda. We also have obtained the 28-aa synthetic peptide, and are exploring the possibility that it modulates ENase subunit association. While this peptide has no detectable effect on dimeric PvuII ENase, it inhibits renaturation of urea-denatured ENase in a concentration-dependent manner. The ORF may represent an additional safeguard during establishment of the PvuII restriction-modification system in a new host cell, helping to delay the appearance of active ENase dimers, while the MTase accumulates and protects the host chromosome.
已发现PvuII限制修饰系统包含三个基因，分别编码一种DNA甲基转移酶（MTase）、一种限制内切酶（ENase）以及ENase编码基因表达所需的一种小蛋白。此外，在MTase编码基因内部及相对位置存在一个小的开放阅读框（ORF）。包含该ORF的区域会被转录，且该ORF具有优良的Shine-Dalgarno序列及ATA起始密码子。在SmaI系统中存在一个与之密切相关的ORF。来自PvuII ORF的预测的28个氨基酸（aa）的肽与PvuII ENase在二聚体界面处的一个区域相似。我们已克隆了这个ORF，赋予其ATG起始密码子，并将其置于诱导型启动子的控制之下：诱导会导致噬菌体λ的限制作用出现轻微但显著的降低。我们还获得了该28个氨基酸的合成肽，并正在探索其调节ENase亚基缔合的可能性。虽然该肽对二聚体PvuII ENase没有可检测到的影响，但它以浓度依赖的方式抑制尿素变性的ENase的复性。该ORF可能代表了在新宿主细胞中建立PvuII限制修饰系统过程中的一种额外保障，有助于延缓活性ENase二聚体的出现，同时MTase积累并保护宿主染色体。
