Award details

Mechanisms of transcription termination

Reference	BB/L010003/1
Principal Investigator / Supervisor	Professor Nikolay Zenkin
Co-Investigators / Co-Supervisors
Institution	Newcastle University
Department	Inst for Cell and Molecular Biosciences
Funding type	Research
Value (£)	358,304
Status	Completed
Type	Research Grant
Start date	10/12/2013
End date	09/12/2016
Duration	36 months

Abstract

Our overall goal is to understand the mechanisms of termination by archaeal RNA polymerase and eukaryotic RNA polymerase I. Most of the studies of transcription termination focused on efficiency of pausing at the termination site, while mechanisms of destruction of the paused complexes (i.e. release of RNAP and RNA from template DNA) mostly remained overlooked. Recently we discovered an elegant scenario of termination by RNA polymerase III (pol III), in which release of unusual elongation complex formed on termination site is facilitated by folding of the nascent transcript. This work accumulated unique set of experimental tools and became an inspiration for analysis of termination by archaeal RNAP (from T. kodakarensis) and eukaryotic RNA polymerase I, pol I (from S. cereviseae). Our preliminary data revealed that both RNA polymerases cannot dissociate from the template upon reaching the termination signal, as was suggested earlier. In the proposed study we will analyse the structures and characteristics of elongation complexes of archaeal RNAP and pol I, paused at their respective termination sites. We will test the suggested by preliminary data involvement of RNA secondary structures and possible involvement of proteins in the process of termination. We will investigate the possibility of "torpedo" mechanism of termination by pol I, and, if found to be involved, we will investigate in vitro the molecular mechanisms of this path of termination, which is also utilised by pol II but is poorly understood. Also, as suggested by our findings with pol III, the study will likely unveil new and/or unique features of archaeal RNAP and pol I, as well as improve understanding of evolutionary relations among the mechanisms of multi-subunit RNAPs functioning. The proposed involvement of RNA secondary structure in process of termination may unify mechanisms of termination among bacterial, archaeal and eukaryotic RNA polymerases.

Summary

Understanding the molecular mechanisms governing the expression of genes is an essential part of developing strategies for intelligent manipulation of harmful and useful organisms, development of new antimicrobial and antifungal agents, fighting diseases, and, importantly, for understanding emergence and evolution of Life. In all living organisms the first step of gene expression is accomplished by highly conserved multi-subunit RNA polymerases (RNAPs). Termination is an essential step of transcription that ensures recycling of RNAP and proper expression of neighbouring genes. However, the mechanisms of termination remain poorly understood. Recently we discovered an elegant scenario of termination by RNA polymerase III, which transcribes many essential genes in eukaryotic cell. We discovered analogies with bacterial termination, which suggest that this fundamental mechanism have emerged billions years ago, before divergence of three domains of life. This research inspired us to investigate the mechanisms of termination by RNA polymerase I, which synthesises most of RNA in eukaryotic cell, and Archaeal RNAP, which functions in the third, much understudied, domain of life. Malfunctions in RNA polymerase I is linked with diseases such as cancer, Alzheimer's and others. Archaea are important players in ecology and biotechnology, as well as inhabit humans and ruminant's guts. Therefore besides fundamental importance, the proposed study may have an impact on medical and environmental research, agriculture and biotechnology. Also, as suggested by our findings with RNA polymerase III, the study may reveal new and/or unique features of these RNA polymerases as well as improve understanding of evolutionary relations among the mechanisms of multi-subunit RNAPs' functioning. The wide range of techniques required to achieve these aims have uniquely accumulated through years in our lab, and for example allowed us to decipher mechanism of termination by RNA polymerase III, describe a mechanism involved in regulation of the development of HIV-1, and to uncover mode of action of antibiotic Tagetitoxin. The proposed research will add to these techniques and will provide new tools for investigation of the first step of gene expression as well as for design and testing of novel drugs.

Impact Summary

Academia: >International scientific community: Transcription is among the most fundamental and evolutionary conserved biological machineries. The similarities or differences in mechanisms of transcription termination in eukaryotes and archaea will provide new insights into the evolutionary relations between these taxa. >Molecular biology: The proposed research will provide new insights into molecular mechanisms of functioning of RNA polymerases. Addressing questions of the proposed study may undermine dogmatic views on some of the features of eukaryotic and archaeal transcription machineries. >Diseases research: Malfunction of pol I and pol III were linked with diseases such as cancer, Alzheimer's, heart diseases, multiple sclerosis and others. Investigation of the mechanism of termination of these RNA polymerases is therefore crucial for understanding of regulation of transcription and possible malfunctions during diseases development. >Biophysics: The possible unusual conformations of paused elongation complexes of RNA polymerases (for example the one that we found for pol III), will be of interest for structural biologists, such as crystallographers, cryo-electron and atomic force microscopists. >Synthetic biology: Archaea are important for bioengineering and biotechnology. The proposed research will shed light on the mechanisms governing the first step of gene expression in Archaea. The pause sequences and termination processes elucidated in our study may be applied in intelligent manipulation of gene expression. UK scientific competitiveness: The scientific disciplines mentioned above are very competitive internationally. Furthermore, the arsenal of methodology designed in this study is unique. The research will also attract good scientists from abroad. These factors will strengthen the scientific competitiveness of the UK. Commercial private sector: The University based bio-tech company (mentioned in the Pathway to Impact) expressed considerable interestin our work. They possess a large library of antibiotics, potentially novel inhibitors of RNAP, and are interested in testing these inhibitors in our experimental systems (a letter of support is available upon request). The results with archaeal RNA polymerase may attract interest from biotechnology companies working on development of Archaea-produced enzymes or biofuels or from agricultural companies working with ruminants. Wider public: Given that the mechanisms of elongation are highly conserved, our results will be useful for understanding and, as a result, for intelligent manipulation of fungi and Archaea. Proposed research addresses (i) mechanisms governing functioning of pol I, whose malfunction was linked with diseases such as cancer, Alzheimer's, multiple sclerosis, heart diseases and others; and (ii) transcription in Archaea, important in environment, agriculture and biotechnology. Therefore, potential long term beneficiaries will be health and environmental organisations, biotechnology and consequently the wider public. Methods designed during the proposed study will provide additional tools for development of antibacterials and antifungals acting against transcription. We already applied some tools, partly developed during preliminary work, to elucidate the mode of action of an inhibitor of RNAP, antibiotic Tagetitoxin. Furthermore, we collaborate with a bio-tech company (see above), who provide us with a library of novel inhibitors of transcription, investigation of which will benefit from the experimental tools designed in the proposed study. Additional potential benefits for the "wider public" will be in publicising the research via press releases, interviews, etc. The Impact activities will be managed by the PI and the collaborators. In the Newcastle University it will be supported by NU commercial development team and press office that are partly funded via this grant.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Structural Biology
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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