HDV RNA Folding and PKR Protein Regulation

描述(由申请人提供)： RNA是多种生物过程的中心，包括转录、剪接、翻译、基因表达、发育和细胞分裂。因此，了解RNA如何折叠到执行这些功能所需的结构中是很有意义的。同样重要的是要了解天然RNA和RNA折叠中间体的结构如何调节关键的生物学过程，包括干扰素诱导的抗病毒制剂蛋白激酶PKR的激活。这一建议涉及研究转录过程中发生的RNA折叠事件，并了解它们与添加二价离子时发生的折叠事件的异同。我们将研究转录暂停与真实模板和聚合酶结合的重要性。此外，还将研究非天然或替代配对抑制并在特定情况下刺激丁型肝炎病毒(HDV)催化RNA折叠的能力。将系统地研究外部因素，如离子强度增加和丁型肝炎抗原蛋白(HDAg)对替代配对拆分的影响。这些RNA折叠状态激活PKR的程度也将被研究。最近发现的一个新的激活PKR的小RNA基序将被机械地研究。这些研究将使用生化和生物物理技术进行，包括PKR激活和快速猝灭RNA裂解动力学；停流荧光和吸收动力学；热力学测量；以及RNA结构图谱。 预计这些结果可能会对与人类健康相关的几个领域产生影响，包括了解HDV的复制，这会增加乙肝病毒(HBV)感染的毒力，以及调节PKR蛋白，PKR蛋白是人类病毒防御机制的一部分。此外，这些研究可能有助于揭示该激酶在体内的新作用。预计这一结果将对RNA折叠社区产生根本的影响，并可能影响对其他生物相关RNA和RNA-蛋白质系统中RNA折叠的理解，包括催化RNA和核糖体。

DESCRIPTION (provided by applicant): RNA is central to a variety of biological processes including transcription, splicing, translation, gene expression, development, and cell division. It is therefore of interest to understand how RNA folds into the structures necessary to carry out these functions. It is also important to understand how the structures of native RNA and RNA folding intermediates regulate critical biological processes including activation of the interferon-induced anti-viral agent protein kinase PKR. This proposal involves studying RNA folding events that occur during transcription, and understanding their similarities and differences to refolding events that occur upon addition of divalent ions. The importance of transcriptional pausing with the authentic template and polymerase will be investigated. In addition, the ability of non-native, or alternative pairings, to inhibit and, in selected cases, stimulate the folding of the catalytic RNA from hepatitis delta virus (HDV) will be studied. The influence of external factors, such as increased ionic strength and hepatitis delta antigen protein (HDAg), on resolution of alternative pairings will be systematically investigated. The extent to which these RNA folding states can activate PKR will be investigated as well. A recently discovered novel small RNA motif that activates PKR will be studied mechanistically. These studies will be carried out with biochemical and biophysical techniques, including PKR activation and rapid-quench RNA cleavage kinetics; stopped-flow fluorescence and absorbance kinetics; thermodynamic measurements; and RNA structure mapping. It is anticipated that these results may impact upon several areas relevant to human health including understanding replication of HDV, which increases the virulence of hepatitis B virus (HBV) infections, and regulation of PKR protein, which mediates part of the human viral defense mechanism. In addition, these studies may help uncover new roles for the kinase in vivo. Results are expected to be of fundamental interest to the RNA folding community and may impact the understanding of RNA folding in other biologically relevant RNA and RNA-protein systems including catalytic RNAs and the ribosome.