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Transcription elongation processivity: backtracked complexes formation and resolution

Reference	BB/F013558/1
Principal Investigator / Supervisor	Professor Nikolay Zenkin
Co-Investigators / Co-Supervisors
Institution	Newcastle University
Department	Inst for Cell and Molecular Biosciences
Funding type	Research
Value (£)	387,139
Status	Completed
Type	Research Grant
Start date	11/08/2008
End date	10/08/2011
Duration	36 months

Abstract

Transcription elongation is frequently interrupted by signals that result in backtracking of the elongation complex, and thus in its inactivation. Backtracked complexes can be reactivated by hydrolysis of the transcript in the RNA polymerase active centre. However, current knowledge does not explain how backtracked complexes are reactivated, since the cleavage reaction is thought to be too slow, and cleavage factors were shown to be dispensable for cells. We are going to investigate the mechanisms of formation of, and rescue from backtracked elongation complexes, which are poorly understood. We will test the hypothesis, emerging from my preliminary results, that the resolution of backtracked complexes by transcript hydrolysis takes place at specific sites on the DNA template, where transcript hydrolysis is highly increased. We will characterise these sites and describe mechanisms of cleavage activity acceleration at them, and the mechanisms of their recognition by backtracking RNA polymerase. We will also test the hypothesis, based on my recent discovery of the transcript-assisted RNA cleavage, that the transcript participates in the resolution of the backtracked complex by providing active groups to the active centre of RNA polymerase and activating phosphodiester bond hydrolysis. We will investigate the mechanisms of transcript induced activation of the reaction. Using novel techniques that we have developed, we will investigate the preferences of elongating RNA polymerase for DNA and RNA sequences, so as to understand the mechanisms of backtracking, backtracked complex rescue, various kinds of elongation pauses, and to improve understanding of structure of elongation complex.

Summary

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.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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