Abe H, Aiba H
Department of Molecular Biology, School of Science, Nagaya University, Japan.
Biochimie. 1996;78(11-12):1035-42. doi: 10.1016/s0300-9084(97)86727-2.
The hallmark features of rho-independent transcription terminators are a G(+)C-rich dyad symmetry sequence followed by a run of T residues on a sense strand. Both of these structural elements are required for efficient transcription termination. Besides its primary function, rho-independent terminators are also known to enhance expression of an upstream gene by stabilizing RNA in a few cases. The Escherichia coli crp gene encoding cAMP receptor protein (CRP) contains a typical rho-independent terminator. To gain further insight into the roles of the G(+)C-rich dyad symmetry sequence and the poly(T) tract both in transcription termination and mRNA stabilization, we constructed a series of variant crp terminators and analyzed their abilities regarding these two functions. Disruption of the G(+)C-rich dyad symmetry sequence almost completely eliminated terminator activity while disruption of the poly(T) tract reduced terminator activity significantly but not completely. Thus, the contribution of the G(+)C-rich dyad symmetry sequence to transcription termination is larger than that of the poly(T) tract. Disruption of the G(+)C-rich dyad symmetry region reduced expression of the upstream crp gene by accelerating the rate of mRNA degradation. However, disruption of the poly(T) sequence had no effect on the stability of the crp mRNA, indicating that the poly(T) tract plays no role in mRNA stabilization. When the crp terminator was replaced by terminators derived from other genes, the fusion genes expressed the crp mRNA at the same level as did the native crp gene, suggesting that the mRNA stabilization effect is probably a general nature of rho-independent terminators.
不依赖ρ因子的转录终止子的标志性特征是富含G（+）C的二元对称序列，随后是有义链上的一串T残基。这两个结构元件都是高效转录终止所必需的。除了其主要功能外，在少数情况下，不依赖ρ因子的终止子还通过稳定RNA来增强上游基因的表达。编码cAMP受体蛋白（CRP）的大肠杆菌crp基因包含一个典型的不依赖ρ因子的终止子。为了进一步深入了解富含G（+）C的二元对称序列和多聚T序列在转录终止和mRNA稳定中的作用，我们构建了一系列变体crp终止子，并分析了它们在这两种功能方面的能力。富含G（+）C的二元对称序列的破坏几乎完全消除了终止子活性，而多聚T序列的破坏显著降低了终止子活性，但没有完全消除。因此，富含G（+）C的二元对称序列对转录终止的贡献大于多聚T序列。富含G（+）C的二元对称区域的破坏通过加速mRNA降解速率降低了上游crp基因的表达。然而，多聚T序列的破坏对crp mRNA的稳定性没有影响，这表明多聚T序列在mRNA稳定中不起作用。当crp终止子被其他基因的终止子取代时，融合基因表达crp mRNA的水平与天然crp基因相同，这表明mRNA稳定作用可能是不依赖ρ因子的终止子的普遍特性。
