Mechanism of Transcription Termination by E. coli RNA Po

本课题主要研究高纯度体外转录系统中大肠杆菌和酿酒酵母的转录终止机制。该系统包括大肠杆菌RNA聚合酶[β‘亚基(RpoC)心型末端标记的六组氨酸]和酵母RNA聚合酶II(Pol II，标记在RPB3亚基N端的六组氨酸)核心酶固定在固相中，将合成的DNA和RNA组分添加到其中以重建真正的延长复合体。延长复合体代表一种形式的RNA聚合酶，它沿着基因行进，合成RNA转录本。正常情况下，伸长复合体高度稳定，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，“抓地力”就会解除，复合体就会瞬间解体。该终止子由两个元件组成：在RNA聚合酶后面的RNA中形成稳定的发夹状结构，以及位于RNA中发夹旁边的寡核苷酸轨迹。导致RNA聚合酶终止子解离的机制尚不清楚。人们认为，RNA末端与移动聚合酶中的DNA杂交的强度是延长复合体稳定性的主要因素。当杂交体较弱或较短时，复合体自发解离。发夹可能的工作原理之一是，它会使聚合酶中的RNA：DNA杂交减少到超出正常长度的程度。我们通过对伸长复合体在不同转录终止子变体上的解离动力学的统计测量，讨论了RNA发夹在伸长复合体失稳中的作用。该项目包括四个连续的步骤。首先对伸长复合体进行重组，然后用盐敏实验对其稳定性进行分析。我们所达成的结论可概括如下： (I)我们发现发夹的折叠破坏了8-bp RNA：DNA杂交的三个上游碱基对，这是延伸复合体中的一个主要稳定性决定因素。在终止过程中，发夹不会直接与8个碱基的杂交序列竞争碱基配对。因此，杂交种的融化是由于RNA聚合酶的空间限制，它将发夹的形成与紧邻茎下游的杂交种的破坏耦合在一起。将弱的RU：DA杂交体从8个核苷酸缩短到5个核苷酸会导致络合物解离。 (Ii)我们证明了一种类似的机制，涉及8-碱基RNA：DNA杂交的发夹熔化，在体外破坏酵母RNA聚合酶II的延伸复合体。 因此，在原核生物和真核生物中，RNA：DNA杂交体的融化作为诱导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.