Mechanism of Transcription Termination by E. coli RNA Po

本项目主要研究大肠杆菌和酵母S.酿酒酵母在高度纯化的体外转录系统。该系统涉及E. coli RNA聚合酶[在β ′亚基（rpoC）的碳末端标记六组氨酸]和酵母RNA聚合酶II（Pol II，在RPB 3亚基的N末端标记六组氨酸）核心酶固定在固相中，向其中加入合成的DNA和RNA组分以重建真实的延伸复合物。延伸复合物代表RNA聚合酶的一种形式，其沿着基因行进并合成RNA转录物。通常，延伸复合物是高度稳定的，并且非常紧密地抓住DNA和RNA。然而，当RNA聚合酶到达称为转录终止子的序列时，“抓持”被解除，复合物福尔斯瞬间分开。终止子由两个元件组成：在RNA聚合酶后面的RNA中形成的稳定的发夹样结构，以及位于RNA中发夹旁边的寡尿苷轨道。导致RNA聚合酶在终止子处解离的机制尚不清楚。据信，RNA末端与移动的聚合酶中的DNA杂交的强度是延伸复合物稳定性的主要因素。当杂化体弱或短时，复合物自发地解离。发夹如何工作的一个想法是，它减少了聚合酶中RNA：DNA杂交体的正常长度。我们通过对不同转录终止子变体处的延伸复合物的解离动力学进行统计测量，解决了RNA发夹在延伸复合物不稳定中的作用。该项目包括四个连续步骤。首先，重建延伸复合物，然后使用盐敏感性测定分析其稳定性。我们得出的结论可归纳如下： (i)我们发现，折叠的发夹破坏了3个上游碱基对的8-bp的RNA：DNA杂交，一个主要的稳定性决定因素的延长复合物。在终止过程中，发夹不直接与8-bp杂交体竞争碱基配对。因此，杂交体的解链是由RNA聚合酶的空间限制引起的，RNA聚合酶将发夹的形成与杂交体在茎下游的破坏相结合。将弱rU：dA杂合体从8 nt缩短至5 nt导致复合物解离。 (ii)我们表明，一个类似的机制，涉及8 bp的RNA：DNA杂交的发夹融化，破坏酵母RNA聚合酶II在体外的延伸复合物。 因此，RNA：DNA杂合体的解链作为诱导原核生物和真核生物中RNA聚合酶释放的一般机制而出现。

This project concerns the investigation of the mechanism of transcription termination in Escherichia coli and the yeast S. cerevisiae in the highly purified in vitro transcription system. This system involves the E. coli RNA polymerase [hexahistidine-tagged at the carboy-terminus of beta' subunit (rpoC)] and the yeast RNA polymerase II (Pol II, hexahistidine-tagged at N-terminus of RPB3 subunit) core enzyme immobilized in a solid phase to which synthetic DNA and RNA components were added to reconstruct the authentic elongation complex. The elongation complex represents a form of RNA polymerase traveling along the gene and synthesizing the RNA transcript. Normally, the elongation complex is highly stable and grips the DNA and the RNA very tightly. However, when RNA polymerase reaches the sequence called transcription terminator, the "grip" is relieved and the complex falls apart momentarily. The terminator consists of two elements: the stable hairpin-like structure formed in the RNA behind RNA polymerase, and the oligo-uridine track located next to the hairpin in the RNA. The mechanism, that causes RNA polymerase dissociation at terminator is unknown. It's believed, that the strength with which the end of the RNA is hybridized to the DNA in the moving polymerase is a major factor of the elongation complex stability. When the hybrid is weak or short the complex spontaneously dissociates. One of the ideas how the hairpin might work is that it reduces the RNA:DNA hybrid in the polymerase beyond its normal length. We addressed the role of the RNA hairpin in destabilization of elongation complex by performing the statistical measurements of the dissociation kinetics of elongation complexes at different variants of transcription terminator. The project involved four consecutive steps. First, the elongation complex was reconstituted followed by the analyses of its stability using the salt-sensitivity assay. The conclusions we have reached can be summaried as follows: (i) We show that folding of the hairpin disrupts the three upstream base pairs of the 8-bp RNA:DNA hybrid, a major stability determinant in the elongation complex. During termination, the hairpin does not directly compete for base pairing with the 8-bp hybrid. Thus, melting of the hybrid results from spatial restrictions in RNA polymerase that couple the hairpin formation with the disruption of the hybrid immediately downstream from the stem. Shortening the weak rU:dA hybrid from 8 nt to 5 nt causes dissociation of the complex. (ii) We demonstrate that a similar mechanism, involving melting of 8-bp RNA:DNA hybrid by the hairpin, disrupts elongation complexes of yeast RNA polymerase II in vitro. Thus, melting of the RNA:DNA hybrid arises as a general mechanism inducing RNA polymerase release in prokaryotes and eukaryotes.