Regulation of elongation by RNA polymerase and ribosome via intrinsic signals and transcription-translation coupling

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


Understanding the molecular mechanisms governing the expression of genes in the cell is an essential part of developing strategies for intelligent manipulation of harmful and useful organisms, development of new antimicrobial and antifungal agents, and, importantly, for understanding emergence and evolution of Life.
In bacteria, both steps of gene expression, copying genes into RNA (transcription) and production of proteins on RNA (translation), are coupled. This implies tight interactions and mutual regulation of these two molecular machineries, which determine the overall rate of gene expression, which, in turn, is important for all cellular processes. This regulation and mechanisms determining the rates of transcription and translation are poorly understood.
We are the first laboratory who developed a unique experimental system where both steps of gene expression can be assembled from purified components and investigated simultaneously in the test tube. We also possess some unique biochemical techniques, which allowed us for example to describe the mechanism involved in regulation of the development of HIV-1, and to uncover mode of action of antibiotic Tagetitoxin. In this proposal, by using these techniques, we will investigate what factors determine rates of both steps of gene expression: (i) we will apply a systematic approach to understand the signals in the DNA that may influence the rate of transcription; (ii) we will investigate the interactions and mutual regulation of transcription and translation.
Sophisticated regulation of transcription and translation are the major factors for survival of pathogenic bacteria. Understanding of relations between these two steps of gene expression will improve our knowledge of these bacteria, and thus provide new tools for fighting infectious diseases. We collaborate with a bio-tech company on applying experimental tools developed in our research to elucidation of modes of action of new antibiotics, inhibitors of RNA polymerase. Being at the interface of transcription and translation, the proposed study will be valuable for both of these vast fields of molecular biology. We collaborate with the leading UK laboratories who will use the tools designed in this study to investigate mechanisms of gene expression by biophysical methods, such as single-molecule techniques, crystallography, cryo-electron microscopy. Furthermore, simultaneous assembly of the two major cellular machineries in the test tube may become the first step for production of artificially assembled living organisms.

Technical Summary

Our overall goal is to understand the rules that determine the rate of expression of a gene.
Transcription elongation is now recognized as one of the major targets of regulation of gene expression in eukaryotes and prokaryotes. The mechanisms governing pausing and determining the overall rate of transcription elongation are poorly understood. In the proposed research we will test the hypothesis, emerged from our preliminary results, that recognition of nucleic acids sequences in the elongation complex, which causes translocation pausing, determines the overall rate of transcription elongation. We will elucidate the rule of sequence recognition by RNA polymerase during elongation. By using selection in vitro, SELEX, we will perform a robust search for signals that may cause pausing of transcription. This will be the first systematic approach to investigation of transcription elongation and the obtained data will be used to build a comprehensive mathematical model of transcription elongation and pausing.
Besides intrinsic signals, the elongation rate and pausing by RNAP is also influenced by translation which, in bacteria, is coupled to transcription. These relations between the two machineries remain unclear, mostly due to the lack of an in vitro experimental system. In the proposed study, we will use the first in vitro coupled transcription-translation system developed by us. We will measure the distances between ribosome and RNA polymerase. We will analyze the effects of transcription-translation coupling on processivity of transcription, i.e. on known and newly described pauses of transcription, and on transcription translocation. Preliminary results suggest that transcription pausing may regulate the rate of translation, which may be important for protein folding, or incorporation of proteins into the membrane. We will analyze if known and newly described transcriptional pauses may regulate translation by pausing ribosome.

Planned Impact

>International scientific community: Transcription and translation are highly conserved in all living organisms and are important targets for regulation. The knowledge obtained in the proposed research will be of importance for researchers from vast fields of transcription and translation (in all three domains of life), as well as to scientists working at their interface.
>Systems biology: The proposed research involves first experimental-based systematic investigation of sequence-dependency of transcription elongation and pausing. We collaborate with a theoretical biophysicist who will be producing mathematical modelling based on our results (letter of support is attached). These data will be important for systems approach of studying gene expression.
>Biophysics: The sequences that freeze thermal motion of RNAP along the template and coupled transcription-translation system, developed in our study, will be of practical importance for single-molecule, crystallographic and/or cryo-EM studies. We collaborate with UK leaders in single-molecule techniques and Cryo-EM studies (letters of support available upon request).
>Synthetic biology: In this proposal we assemble the first in vitro system, which involves both steps of gene expression, transcription and translation. This may become a valuable instrument for biosynthetic systems, as well as the first step for development of an artificial life from purified components. The pause sequences 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 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)
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 pathogenic bacteria and fungi. Proposed research may lead to novel antimicrobial targets. Therefore, potential long term beneficiaries will be health organisations and consequently the wider public.
The growing resistance of pathogenic bacteria to existing antibiotics requires the invention of new antimicrobial agents. RNAP has been validated as a target for antibiotics by the clinical use of Rifampicins. A new RNAP inhibitor, Lipiarmycin (Fidaxomicicn), is currently undergoing phase III clinical development. Understanding the mechanism of action of newly discovered inhibitors is an essential step in their development, and their intelligent improvement. Methods designed during the proposed study will provide additional tools for development of antibacterials and antifungals acting against transcription. We already applied these 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.


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Description Transcription translation coupling:
We have discovered that two most important gene expression processes in the bacterial cell, transcription and translation, that work together are much more tightly interact with each other than previously thought. We met the objective and measured the distance between the coupled ribosome and RNA polymerase (unpublished, NAR, Methods, Methods in molecular biology).

Transcription regulation through pauses and fidelity of elongation:
We confirmed the existence of pre-translocated pauses, and found that antibiotic Tagetitoxin specifically targets such pauses, slowing down RNAP progression even further. Tagetitoxin is the first antibiotic to solely target translocation (NAR). We hypothesised in the proposal that pre-translocation pauses are ubiquitous and attempted to determine the sequences that cause these pausing biochemically. However newly developed techniques based on next generation sequencing proved far more powerful. We therefore have re-directed our attempts towards these methods. So far we produced pipelines for analysis of deep sequencing data to determine transcription pausing and fidelity in vivo. We also developed technique revealing pausing sites (Micrococcal nuclease (MN)-seq; unpublished), and started setting NET-seq and ribosome profiling (developed elsewhere).
Based on extensive biochemical, microbiological characterisation and mathematical modelling we proposed that the major role of transcription factor Gre in Streptococcus pneumonia is to prevent traffic jams of RNAPs caused by transcription pausing and misincorporation by the leading RNAP (NAR).
We discovered the role of DksA/ppGpp in fidelity of transcription, which possibly explains the long standing mystery of their involvement in resolution of conflicts between transcription and replication. We suggest they act by increasing fidelity of transcription and thus preventing misincorporation pauses (NAR).
We found that a transcription factor (p7 protein of bacteriophage Xp10 of the major rice pathogen X. oryzae) can increase processivity of transcription elongation by "pushing" multi-subunit RNAP forward thus suppressing various types of pauses (NAR).

Other findings related to the project:
We found that antibiotic Streptolydigin requires non-catalytic Magnesium ion for binding to RNAP, the first known example of involvement of non-catalytic metal ions in antibiotic action (Antimicrobial agents and chemotherapy).
We found that eukaryotic RNAP II uses transcript assisted proofreading mechanism, when RNA helps to correct itself, as do bacterial RNAPs (Transcription).
We discovered that termination by eukaryotic RNAP III, that synthesises the most number of RNAs in the cell, involves backtracking and the secondary structure of the transcript (Science).

Using developed by us translation in vitro system:
We uncovered a molecular mechanism for the main bacterial persistence (temporary antibiotic resistance) protein, toxin HipA (MolCell), which we showed phosphorylates Glu-tRNA-synthetase. We also determined the mode of actionof another bacterial toxin Doc; we showed that it phosphorylates essential translation factor EF-Tu, the finding that revealed a new way of evolution of novel catalytic activities (NatureChemBiol).

We have shown that ribosome can physically push RNAP from the arrested state, thus explaining how translation can preclude conflicts of transcription and replication
Exploitation Route Our findings on transcription factors and action of Tagetitoxin will be of use in understanding the mechanisms of transcription pausing and conflicts of transcription with replication and be useful controls in global investigations of pausing and conflicts in vivo.
Micrococcal nuclease (MN)-seq developed to assess pausing in vivo appears to be cheaper and simpler than an alternative NET-seq (though the resolution of MN-seq is lower), and could be used for simpler applications where high resolution of data is not required.
The coupled transcription translation system will be used (and is already applied by our collaborators) for crystallographic and Cryo-EM studies. It also will be useful for synthetic biology as one of the most complex biological systems assembled from purified components.
The findings of modes of action of toxins HipA and Doc will be of use in studying bacterial persistence and possible roles of native phosphorylation of EF-Tu.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description This was a fundamental study into transcription pausing and coupling of transcription and translation, and the immediate impact on society and economy was not expected. However, as an indirect impact on economy from the project was the usage of the experimental systems developed during the study by our collaborators biotech company Demuris Ltd in search for novel antibiotics targeting transcription and translation. We expect that the impact on the scientific community by our study and related projects (particularly coupled transcription-translation and discoveries made using in vitro translation) will bring societal and economic impacts in the future, providing tools for drug discovery and synthetic biology/biotechnology. Our findings on regulation of transcription of X. oryzae by a bacteriophage protein could be of importance in attempts of fighting this major rice pathogen. The finding on the mode of action of Tagetitoxin and on mode of binding of Streptolydigin may be of use in future drug development. Findings on bacterial toxins HipA and Doc and their involvement in bacterial persistence could also be of interest for pharma and biotechnology.
First Year Of Impact 2015
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

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