Looman A C, Bodlaender J, de Gruyter M, Vogelaar A, van Knippenberg P H
Nucleic Acids Res. 1986 Jul 11;14(13):5481-97. doi: 10.1093/nar/14.13.5481.
Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E. coli and then studied the variation in expression of the fusions. The RBSs originated from bacteriophage Q beta and MS2 genes and the E. coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK). The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold. Lac-specific mRNA synthesis follows the variation in protein production. It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon. This efficiency is context dependent. The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results. Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.
我们使用先前描述的载体（pKL203），将几个异源核糖体结合位点（RBS）与大肠杆菌的lacZ基因融合，然后研究融合体表达的变化。这些RBS来源于噬菌体Qβ和MS2基因以及大肠杆菌中延伸因子EF-Tu A和B和核糖体蛋白L11（rplK）的基因。通过免疫沉淀法测定lacZ融合蛋白的合成，发现其变化至少达100倍。Lac特异性mRNA的合成随蛋白质产生的变化而变化。似乎RBS在融合基因表达中发挥作用的效率与缺乏涉及Shine和Dalgarno核苷酸（SDnts）和/或起始密码子的二级结构之间存在相关性。这种效率取决于上下文。单独的SD nts序列以及直至起始密码子的间隔区的长度和序列无法解释我们的结果。在噬菌体Qβ复制酶RBS中产生的缺失突变揭示了一种复杂的表达控制模式，可能涉及使用“假”起始位点。
