Transcription elongation processivity: backtracked complexes formation and resolution

Lead Research Organisation: Newcastle University
Department Name: Inst for Cell and Molecular Biosciences

Abstract

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.

Technical Summary

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.

Publications

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Nielsen S (2013) Mechanism of eukaryotic RNA polymerase III transcription termination. in Science (New York, N.Y.)

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Yuzenkova Y (2012) Multiple active centers of multi-subunit RNA polymerases. in Transcription

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Yuzenkova Y (2010) Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis. in Proceedings of the National Academy of Sciences of the United States of America

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Yuzenkova Y (2010) Stepwise mechanism for transcription fidelity. in BMC biology

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Zenkin N (2014) Multiple personalities of the RNA polymerase active centre. in Microbiology (Reading, England)

 
Description 1. We discovered an ability of RNA polymerase to recognise sequences of the RNA-DNA hybrid, which can influence the elongation rate via pausing, and showed its involvement in some important transcriptional pauses, for example involved in development of HIV-1 or virulence of some bacteria (EMBOJ).
2. We discovered that RNA polymerase core can recognise upstream DNA sequence during sigma-dependent pausing and discovered a new promoter element recognised by core of RNAP (EMBOJ).
3. We discovered that translocation can be rate limiting step of transcription elongation, thus being the first evidence for "in pathway" pausing (EMBOJ, NAR).
4. We discovered that antibiotic Tagetitoxin inhibits translocation by RNA polymerase, thus being the first known inhibitor targeting solely translocation. This also highlighted the role of the Trigger Loop in translocation and provided an experimental system to analyse translocation (NAR).
5. We discovered that in response to backtracking and/or misincorporation, Gre factor substitutes for the catalytic domain of the RNA polymerase active centre, the Trigger Loop, to switch the activities of the active centre for efficient resolution of these dead end complexes. This is the first example of an exchange of catalytic domains in proteinaceous enzymes (Transcription, Microbiology, NAR).
Taken together the above discoveries brought insights into how RNA polymerase moves along the DNA template during transcription and how this movement can be regulated or inhibited in the case of antibiotics.
Exploitation Route We generated around 30 new plasmids and E. coli strains for overexpression of mutant E. coli and T. aquaticus RNA polymerase and accessory factors.
We have highly improved the technique of assembled elongation complexes, by in depth analysis of the properties of few dozens of various complexes. We found complexes that can change translocation properties of RNA polymerase. The properties of the complexes are published and are used in crystallography, Cryo-EM and single-molecule techniques by our collaborators.
We showed that Tagetitoxin inhibits translocation of elongation complexes, that may now be used as a tool for analysis of phenomenon of translocation.
Sectors Chemicals,Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The PI gave a live phone interview to the radio station BBC Russia in May of 2008. The interview was then deposited on-line on the website of BBC. The interview was in relation to a discovery made by Newcastle University scientists in the area of stem cells research. The PI explained to the audience in lay terms the principles behind stem cell research, the potential benefits and downsides of it. Then, in the conversation, the PI described the functioning of a biochemical/molecular biology laboratory, what an average day of a research associate scientist and a PI looks like. Given that the PI had experience of working in Russia and USA, he explained the benefits of being a young PI and setting up a laboratory in the UK, and funding opportunities in the UK. The PI and the RA were involved in several university open days, and in the Baddiley-Clark Building (where the lab is based) Official Opening by Sir Paul Nurse, FRS, which involved informal communication and visits to the labs by the guests, including the Lord Mayor of Newcastle. The PI participated in the "Patient and Public Engagement: Improving the Impact of Teaching, Learning and Research" conference held by Beacon North East. We have set up a collaboration with a University campus-based biotech company, Demuris Ltd. (www.demuris.co.uk), chaired by Professor Jeff Errington FRS. Demuris are using experimental systems developed in the course of the grant for search of novel antibiotics targeting transcription. Demuris have agreed to supply us with these compounds on a marginal cost basis for investigation of their modes of action. Demuris provide a potential route to clinical practice for inhibitors that turn out to have commercial potential.
Sector Chemicals,Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Societal

 
Description Coupling of transcription with other cellular processes
Amount £2,086,031 (GBP)
Funding ID 217189/Z/19/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 08/2024