Regulation of transcription factor motor activity by autoinhibition and interaction with RNA polymerase

这个项目涉及分子马达的作用和控制，分子马达参与基因的修复和调节。分子马达分解化学燃料(通常是一种叫做三磷酸腺苷的分子)，释放的能量被用来做某种形式的物理工作。这个项目中研究的马达是一种名为MFD的细菌蛋白质，它使用ATP来推动包含细胞遗传信息的双链DNA分子的运动。这是有用的，因为MFD可以将自己附着在另一种称为RNA聚合酶的蛋白质复合体上，当MFD沿着DNA移动时，它会推动RNA聚合酶向前移动。RNA聚合酶是读取DNA中遗传信息的机制的关键部分，MFD对它的影响取决于环境：有时MFD帮助RNA聚合酶开始自己沿着DNA移动，而另一些时候MFD将RNA聚合酶赶出DNA。这些相互作用可以帮助细胞控制基因的使用和维护方式：例如，如果DNA受损，从DNA中移除RNA聚合酶尤其重要，因为RNA聚合酶可以阻止DNA修复，这将增加发生突变或细胞死亡的机会。为了让细胞正常运作，必须仔细控制马达蛋白。如果他们被允许在错误的地点或错误的时间工作，他们可能会干扰电池的其他组件，如果允许他们连续运行，他们将浪费燃料，代价是其他依赖能源的过程。在MFD的情况下，只有当蛋白质与RNA聚合酶结合时，才需要启动运动活性。我们发现，这是由一种类似于电动割草机上的“死人开关”的机械装置来确保的。MFD蛋白的一部分起着开关的作用：当MFD被RNA聚合酶控制时，马达启动，但如果RNA聚合酶松开MFD，马达就会自动关闭。组成这个项目的实验将使我们能够了解MFD中的这种分子开关是如何工作的，以及RNA聚合酶是如何控制它的。这项工作解决了分子生物学中的一个基本问题：在所有类型的细胞中都有许多类似的马达，这些马达通常作为更大的复合体的一部分发挥作用，这些复合体通过未知的机制控制它们的活动。这些马达执行与MFD不同的功能，但有足够的共同特征，可以从实验上可驯化的MFD：RNA聚合酶系统中吸取教训，在试图理解更复杂和更难处理的系统时有用，例如一些与人类健康和疾病有关的含有马达的复合体。通过彻底了解这些蛋白质的功能，我们的目标是有助于增加对疾病的理解和设计新的治疗策略。

This project concerns the action and control of a molecular motor that is involved in the repair and regulation of genes. Molecular motors break down chemical fuel (typically a molecule called ATP) and the energy that is released is utilised to do some form of physical work. The motor studied in this project is a bacterial protein called Mfd, which uses ATP to power movement along the double stranded DNA molecules that contains the cell's genetic information. This is useful because Mfd can attach itself to another protein complex, called RNA polymerase, and when Mfd moves along the DNA it pushes RNA polymerase forward. RNA polymerase is a key part of the machinery that reads the genetic information held in DNA, and the effect that Mfd has on it depends on the circumstances: sometimes Mfd helps RNA polymerase to start moving along the DNA on its own, and other times Mfd pushes RNA polymerase off the DNA. These interactions can help the cell to control the way in which genes are used and maintained: for example, removal of RNA polymerase from DNA is particularly important if the DNA is damaged because RNA polymerase can prevent the DNA being repaired, which will increase the chance that mutations will arise or the cell will die. Motor proteins must be carefully controlled in order for cells to function correctly . If they are allowed to work in the wrong place or at the wrong time they can interfere with other components of the cell, and if they are allowed to run continuously they will use fuel wastefully, at the expense of other energy-dependent processes. In the case of Mfd, it is only necessary for the motor activity to be turned on when the protein is bound to RNA polymerase. We have discovered that this is ensured by a mechanism that resembles the 'dead mans switch' on an electric lawnmower. Part of the Mfd protein acts as a switch: the motor is turned on when Mfd is held by RNA polymerase, but if RNA polymerase lets go of Mfd the motor turns itself off. The experiments that make up this project will enable us understand how this molecular switch within Mfd works, and how RNA polymerase controls it. The work addresses a fundamental question in molecular biology: there are many similar motors within cells of all kinds, and these often function as part of larger complexes that control their activity by undefined mechanisms. These motors perform different functions from Mfd, but share sufficient common features for lessons learnt from the experimentally tractable Mfd:RNA polymerase system to be useful when trying to understand systems that are more complicated and harder to work with, such as some of the motor-containing complexes that are involved in human health and disease. By gaining a thorough understanding of the ways in which such proteins function we aim to contribute to increased understanding of disease and the design of novel therapeutic strategies.