Transcription elongation processivity: backtracked complexes formation and resolution

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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.