Mechanisms of transcription termination
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
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.
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.
Technical Summary
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.
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.
Planned Impact
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 interest in 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.
>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 interest in 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.
Organisations
Publications
Forrest D
(2017)
Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase.
in Nature communications
Gamba P
(2018)
Transcription fidelity and its roles in the cell.
in Current opinion in microbiology
Gamba P
(2017)
A link between transcription fidelity and pausing in vivo.
in Transcription
James K
(2017)
Misincorporation by RNA polymerase is a major source of transcription pausing in vivo.
in Nucleic acids research
James K
(2017)
Deep sequencing approaches for the analysis of prokaryotic transcriptional boundaries and dynamics.
in Methods (San Diego, Calif.)
Nielsen S
(2013)
Transcript assisted phosphodiester bond hydrolysis by eukaryotic RNA polymerase II.
in Transcription
Nielsen S
(2014)
Transcription. Response to Comment on "Mechanism of eukaryotic RNA polymerase III transcription termination".
in Science (New York, N.Y.)
Van Nues RW
(2016)
Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate.
in Nucleic acids research
Zenkin N
(2014)
Ancient RNA stems that terminate transcription.
in RNA biology
Zenkin N
(2015)
New Insights into the Functions of Transcription Factors that Bind the RNA Polymerase Secondary Channel.
in Biomolecules
Zenkin N
(2014)
RNA secondary structure-dependent termination of transcription.
in Cell cycle (Georgetown, Tex.)
Zenkin N
(2015)
Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase.
in Nucleic acids research
| Description | 1 (prepared for publication). Our main finding is the discovery of the mechanism of termination by RNA polymerase I (pol I). We developed pol I transcription systems from purified components and within nuclear lysates. We showed that transcription by pol I is stopped by Reb1 road block protein. This is followed by termination that is facilitated by the native RNA hairpin of the nascent pre-rRNA transcript preceding the Reb1 stop site. This RNA hairpin is sufficient and is required for termination. We showed that, upon collision with Reb1, pol I backtracks that allows it to reach the hairpin. Earlier, termination by pol I was believed to take place via "torpedo" mechanism, when, nucleases rnt1 and rat1 degrade the nascent transcript and displace pol I from DNA. We showed that neither rat1 nor rnt1 are needed for efficient termination, and thus the "torpedo" mechanism appears to be redundant. We also showed that no other proteins are required for termination. We suggest that hairpin dependent termination and the other mechanism(s) serve as back-ups to hairpin dependent termination and to each other to ensure the faithful termination by pol I. One of the most significant methodological achievements was setting up transcription by pol I in nuclear lysates, which now can be used by us and other scientists to investigate molecular mechanisms of transcription by pol I. The work led to collaboration with group of Prof Herbert Tschochner from Universität Regensburg, whose lab was visited by the RA to share the experimental experience. 2. Bioinformatics analysis of genomes of several archaea did not reveal any general propensity for stable RNA secondary structures in the vicinity of the ends of the genes and operons. The hairpin dependent termination observed by us in vitro, thus, could be an artefact not relevant to situation in the cell, or, alternatively, be a mechanism specific to particular genes. A relatively weak pausing on polyU signals in vitro also suggests either involvement of additional proteins or DNA topology in the cell, which may also modulate the release of RNA polymerase (RNAP) from the template in the absence of RNA secondary structure. These however are yet to be found/investigated. 3 (NAR). Another significant finding was the discovery of the mechanism of antitermination of transcription by RNAP from the major rice pathogen X. oryzae by bacteriophage protein p7. We showed that p7 promotes forward translocation of RNAP by altering the upstream DNA duplex reformation. This is the second, after NusG, protein regulator that affects translocation of RNAP, indicating that translocation can be a subject of intensive regulation. 4. In side projects partly funded by this grant, (i; NAR) we found an sRNA that binds RNAP, uniquely away from the active centre, and may directly control transcription elongation and determine the fate of the mRNA co-transcriptionally; (ii; under review) we discovered a new type of bacteriophage RNAP, a single-subunit relative of multi-subunit RNAPs; (iii; NAR) we found that misincorporation by RNAP is a major source of transcription pausing and thus collisions with replication in vivo. We described the mechanism of termination by eukaryotic RNA polymerase I |
| Exploitation Route | The impact of this fundamental study is mostly academic. Besides important discoveries mentioned above, we have observed some peculiar features of termination by pol I, which are yet to be investigated by us, our collaborators and other groups. The experimental systems that we use are either unique for pol I research or significantly optimised by us, and will be useful in the future research by us and others. We have established collaboration with group of Prof Herbert Tschochner from Universität Regensburg, whose lab was visited by the RA to share the experimental experience. These experimental systems are planned to be used to analyse interactors/regulators of pol I, that are misregulated in cancers in collaboration with Dr Dr Oxana Bereshchenko from Perugia University. Discovery of sRNA binding RNAP led to collaboration with Prof Ben Luisi from Cambridge University, with whom we try to solve the structure of the complex. Our in vitro systems and discovered by us new RNAP attracted interest from our collaborators bio-tech company Demuris to use them for antibiotic screenings, for heterologous expression of antibiotic cassettes and as a negative control in search for antibiotics targeting bacterial transcription. The unpublished results will be presented at UK RNAP workshop organised by us in Newcastle and at FASEB conference this year. |
| Sectors | Education,Healthcare,Pharmaceuticals and Medical Biotechnology |
| 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 |