Structural Energetics of a RNA Transcription Switch

描述（由申请人提供）：在活细胞中产生蛋白质所必需的遗传信息作为基因储存在DNA中。当基因表达时，这些信息首先被复制到一个互补的信使RNA分子中，这个过程称为转录。活细胞使用各种机制来确保从DNA到信使RNA的信息传递是准确的。一种机制提供了转录应该停止的点的精确定义。这一点必须与基因的末端一致。细胞通过使用特定的终止子位点来定义，策略性地位于基因的末端。在细菌中，大约一半的终止子位点在没有蛋白质帮助的情况下发挥其功能。相反，在这些内在终止子中，转录终止的信号在DNA模板和新生RNA的碱基序列中编码。本申请中提出的研究将使用1H和15 N核磁共振（NMR）光谱来阐明内在终止子位点处转录终止的分子机制。用于研究的靶向核酸分子复制终止子的关键模块。它们包括当转录停止时在新合成的RNA中形成的双螺旋结构，来自转录模板的DNA双螺旋结构，以及将DNA的碱基序列复制到信使RNA中的RNA-DNA杂交结构。这些结构中每个碱基对的稳定性将通过质子-氘分馏因子和亚氨基质子交换速率的NMR测量来定义。研究结果将为每个结构提供高分辨率的能量图。这些图谱将揭示内在终止子的不同模块如何协同工作以实现转录终止所需的功能状态。这些信息将有助于更广泛地理解在正常和患病细胞中表达遗传信息时RNA和DNA中发生的构象转变。

DESCRIPTION (provided by applicant): The genetic information necessary to produce proteins in living cells is stored in DNA as genes. When genes are expressed, this information is first copied into a complementary messenger RNA molecule in a process called transcription. Living cells use various mechanisms to insure that the transmission of information from DNA to messenger RNA is accurate. One mechanism provides the exact definition of the point where transcription should stop. This point must coincide with the end of the gene. The cells make this definition by using specific terminator sites, strategically located at the ends of genes. In bacteria, about half of all terminator sites perform their function without the assistance of proteins. Instead, in these intrinsic terminators, the signal for transcription termination is encoded in the base sequences of the DNA template and of the nascent RNA. The research proposed in this application will use 1H and 15N nuclear magnetic resonance (NMR) spectroscopy to elucidate the molecular mechanism of transcription termination at an intrinsic terminator site. The nucleic acid molecules targeted for investigation reproduce key modules of the terminator. They include the double helical structure that forms in the newly synthesized RNA when transcription stops, DNA double helical structures from the transcription template, and RNA-DNA hybrid structures that copy the sequence of bases from DNA into messenger RNA. The stability of each base pair in these structures will be defined by NMR measurements of protium-deuterium fractionation factors and rates of exchange of imino protons. The results will provide high-resolution energetic maps for each structure. These maps will reveal how different modules of intrinsic terminators work in concert to achieve the functional states necessary for termination of transcription. This information will contribute to the broader understanding of the conformational transitions that occur in RNA and DNA when the genetic information is expressed, in normal and diseased cells.