Rossi J J, Soberon X, Marumoto Y, McMahon J, Itakura K
Proc Natl Acad Sci U S A. 1983 Jun;80(11):3203-7. doi: 10.1073/pnas.80.11.3203.
A prokaryotic consensus sequence promoter has been chemically synthesized and cloned in bacterial plasmid vectors. This designed sequence is biologically active and promotes efficient expression of the genes to which it is fused. It is an unusually strong promoter in vitro, capable of specifying multiple rounds of transcription even when there is a large molar excess of heparin present prior to the addition of RNA polymerase. These properties make this a useful sequence for the in vitro production of RNAs. A 2-base-pair spacer mutant and a -35 region transversion mutant have been created in vitro in the synthetic promoter by synthetic-DNA-mediated, site-specific mutagenesis. The spacer mutant has a marginal in vivo effect on promoter strength but virtually abolishes the in vitro heparin resistance. The -35 region transversion changes a highly conserved nucleotide into the statistically least preferred base. This mutation has no marked effect on in vivo or in vitro promoter strength.
一种原核共有序列启动子已被化学合成并克隆到细菌质粒载体中。这种设计的序列具有生物活性，能促进与其融合的基因高效表达。它在体外是一种异常强大的启动子，即使在添加RNA聚合酶之前存在大量摩尔过量的肝素时，也能够指定多轮转录。这些特性使其成为用于体外生产RNA的有用序列。通过合成DNA介导的位点特异性诱变，在体外合成启动子中创建了一个2碱基对间隔突变体和一个 -35区域颠换突变体。间隔突变体在体内对启动子强度的影响很小，但实际上消除了体外肝素抗性。-35区域颠换将一个高度保守的核苷酸改变为统计学上最不优选的碱基。这种突变对体内或体外启动子强度没有明显影响。
