Transcription elongation processivity: backtracked complexes formation and resolution

转录是基因表达的第一步，也是调控最严格的一步。许多内部和外部信号，如模板中的序列、与模板结合的蛋白质等，都可能影响RNA聚合酶沿DNA的运动。这些信号可以减缓甚至停止转录，从而导致无活性的反向复合体的形成。由于RNA聚合酶沿着DNA模板的反向运动，使RNA的3'端离开活性中心，从而使延伸复合物失活，例如导致转录的过早终止。更重要的是，回溯复合物是对RNA或DNA起作用的不同细胞机器的障碍。因此，细胞应该发明了一种机制来“拯救”或“解决”这些有害的复合物。提出的通过RNA聚合酶活性中心的转录物水解来解决的机制效率太低。增加这种水解活性的分裂因子被证明是细胞不可缺少的。因此，确保有效解决的机制回溯复合物仍然知之甚少。在这里，我提出了一种新的独立于因子的机制来解决回溯复合物，这将解释它们如何在转录过程中有效地解决。该建议基于假设（由初步结果支持）：1)在模板DNA上的特定位点发生回溯复合物的分解，转录物切割反应在此高度增加；Ii)转录本本身协助反向复合体的切割，从而使它们重新激活。我们将测试这些假设，并进一步研究回溯和从中拯救的机制。本研究还将提高对转录延伸复合物的结构和性质的认识，并将阐明转录暂停和终止的机制。

Transcription is the first and the most heavily regulated step of gene expression. Many internal and external signals, such as sequences in the template, proteins bound to the template, etc., may influence the movement of RNA polymerase along DNA. These signals can slow down or even stall transcription, which leads to the formation of inactive backtracked complexes. Backtracked complex forms due to backward movement of RNA polymerase along the DNA template, which leaves the 3' end of the RNA out of the active centre and thus inactivates the elongation complex, e.g. leads to the premature termination of transcription. More importantly backtracked complexes are obstacles for different cellular machineries that work on RNA or DNA. Thus, there should have been mechanisms invented by cells to 'rescue' or 'resolve' these harmful complexes. The proposed mechanism of resolution via unassisted transcript hydrolysis by RNA polymerase active centre was found to be too inefficient. Cleavage factors that increase this hydrolytic activity were shown to be dispensable for cells. Thus, the mechanisms ensuring efficient resolution of backtracked complexes are still poorly understood. Here I propose a new factor-independent mechanism for the resolution of backtracked complexes that would explain how they can be efficiently resolved during transcription. The proposal is based on hypothesises (supported by preliminary results) that: i) resolution of backtracked complexes occurs at specific sites on the template DNA, at which transcript cleavage reactions are highly increased; ii) the transcript itself assists cleavage in the back-tracked complexes, thus reactivating them. We are going to test these hypothesises, and further investigate mechanisms of backtracking and rescue from it. The research of this proposal will also improve understanding of the structure and properties of transcription elongation complexes, and will shed light on the mechanisms of transcriptional pausing and termination.