Zacharias M, Göringer H U, Wagner R
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, FRG.
Nucleic Acids Res. 1990 Nov 11;18(21):6271-5. doi: 10.1093/nar/18.21.6271.
Hybrid promoter constructs were used to determine the DNA sequence requirements for stringent and growth rate control within a promoter region. The promoters were obtained by fusing complementing sequence regions located upstream and downstream from the GCGC discriminator motif of the growth rate regulated rRNA P1 promoter and a non-regulated tac promoter variant. The activities and the regulatory response of the hybrid promoters were determined in vivo using a promoter test vector system with the chloramphenicol acetyltransferase (CAT) reporter gene. Measurements were made at different growth rates and after starvation for isoleucine to induce the stringent response. Neither the upstream nor the downstream sequence of P1 relative to the GCGC discriminator motif conferred comparable regulatory features when fused to the complementing sequences of the non-regulated mutant tac promoter. A minor response to amino acid deprivation or changes in the growth rate was noted for the hybrid promoter with the rrnB P1 upstream segment and the tac downstream element, pointing to a slightly different importance of the two sequence elements for regulation. The parallel effects for stringent as well as growth rate regulation of the hybrid promoters supports the view of a common mechanism for both types of control. However, none of the promoter sequence elements on its own was able to restore the complete regulatory behaviour of their 'parent' promoters.
使用杂交启动子构建体来确定启动子区域内严格控制和生长速率控制所需的DNA序列。这些启动子是通过融合位于生长速率调节的rRNA P1启动子的GCGC鉴别基序上游和下游的互补序列区域以及一个非调节性tac启动子变体而获得的。使用带有氯霉素乙酰转移酶（CAT）报告基因的启动子测试载体系统在体内测定杂交启动子的活性和调节反应。在不同生长速率下以及在异亮氨酸饥饿以诱导严格反应后进行测量。当与非调节性突变tac启动子的互补序列融合时，相对于GCGC鉴别基序的P1的上游或下游序列均未赋予可比的调节特征。对于具有rrnB P1上游片段和tac下游元件的杂交启动子，观察到对氨基酸剥夺或生长速率变化的轻微反应，表明这两个序列元件在调节中的重要性略有不同。杂交启动子对严格控制和生长速率调节的平行作用支持了两种控制类型具有共同机制的观点。然而，没有一个启动子序列元件能够单独恢复其“亲本”启动子的完整调节行为。
