Warburton N, Boseley P G, Porter A G
Nucleic Acids Res. 1983 Sep 10;11(17):5837-54. doi: 10.1093/nar/11.17.5837.
A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E. coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased beta-galactosidase synthesis, were selected for DNA sequencing. One clone has a G leads to A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C leads to T and T leads to C) between the Shine-Dalgarno sequence and initiation codon which raise expression of beta-galactosidase two-fold. A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present. These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.
已开发出一种用于DNA局部诱变的策略。噬菌体M13mp9中的乳糖启动子被大肠杆菌色氨酸启动子取代。仅携带色氨酸启动子区域的一个限制性片段用亚硝酸诱变，重新连接到未诱变的载体上并转染到大肠杆菌中。在指示培养基上产生深蓝色噬菌斑的几个克隆被选出来进行DNA测序，这表明β-半乳糖苷酶的合成增加。一个克隆在“Pribnow框”的3'侧发生了G到A的转换，这导致了一个组成型启动子。两个克隆在Shine-Dalgarno序列和起始密码子之间有不同的点突变（C到T和T到C），这使β-半乳糖苷酶的表达提高了两倍。一个二级结构模型表明，后两个突变可能通过破坏lac Z编码区与核糖体结合位点（RBS）的碱基配对来发挥作用，从而使核糖体更容易接近。该模型的证据来自于以下发现：无论是否存在突变，RBS突变都不会增加与RBS区域没有明显二级结构的不同下游基因的表达。这些结果强化了这样一个假设，即二级结构掩盖是RBS强度的主要决定因素。
