Mechanism of Transcription Termination Protein RHO

大肠杆菌的转录终止蛋白Rho作用于从DNA模板和暂停的RNA聚合酶释放新合成的RNA。Rho是一种只有当RNA结合时才活跃的ATPase，Rho必须水解ATP才能实现转录释放。RNA依赖的核苷酸水解的机制以及这种水解物在转录本释放中的作用尚不清楚。本研究的目的是了解Rho与ATP结合、RNA结合和ATP水解的化学和动力学特征，并将这些知识应用于理解转录本释放的分子机制。建议的实验涉及(1)酶上的化学计量、速率和事件的顺序：有多少分子的配体结合，有多快，以什么顺序；(2)总的反应化学计量：每释放一条RNA链，有多少个ATP分子被水解；以及(3)酶与底物相互作用的分子细节：底物通过什么相互作用结合，哪些氨基酸残基在催化中起重要作用？这些实验测试了Rho作用的特定模型，这些模型将根据结果得到支持或放弃。这项工作的各个方面将有助于理解与ATP或RNA结合的其他蛋白质，以及更普遍的生物能量转导问题。这位研究人员正在研究从信使RNA翻译成蛋白质的过程。这个过程的最后一步是所谓的终止步骤，即完整的蛋白质链从RNA中释放出来。这是一个需要以ATP形式存在的能量的步骤。斯蒂特博士希望阐明这一复杂反应发生的机制。

Transcription termination protein rho of E. coli acts to release newly synthesized RNA from the DNA template and from paused RNA polymerase. Rho is an ATPase which is active only when RNA is bound, and rho must hydrolyze ATP to achieve transcript release. The mechanism of RNA-dependent nucleotide hydrolysis and the role of such hydrolysis in transcript release are not known. The goal of the present research is to understand both chemical and kinetic features of ATP binding, RNA binding, and ATP hydrolysis by rho and to apply this knowledge toward an understanding of the molecular mechanism of transcript release. The proposed experiments are concerned with (1) the stoichiometry, rates, and order of events on the enzyme: how many molecules of ligand bind, how fast, and in what order; (2) the overall reaction stoichiometry: how many molecules of ATP are hydrolyzed per RNA chain released; and (3) the molecular details of interactions between the enzyme and its substrates: what are the interactions by which the substrates bind, and what amino acid residues are important in catalysis? These experiments test specific models of rho action, which will either be supported or discarded based on the results. Aspects of this work will contribute to the understanding of other proteins that bind ATP or RNA and to the more general problem of biological energy transduction. This investigator is studying the process by which proteins are synthesized by the process of translation from messenger RNA. The last step in this process is the so-called termination step in which the completed protein chain is released from the RNA. This is a step which requires energy in the form of ATP. Dr. Stitt wishes to elucidate the mechanism by which this complex reaction takes place.