Structures and Mechanisms of RNA polymerase inhibition and activation

RNA polymerase is a fundamental cellular machinery responsible for converting genetic information stored in DNA to another genetic molecule, called RNA, that can then be converted to protein or act in another regulatory capacity. Accessing information in DNA occurs in a complex, highly controlled process called gene transcription and the core molecular machinery, the RNAP enzyme, is conserved from bacteria to humans. Gene transcription is a highly regulated event in development and a major response to growth and environmental stimuli in all known living systems. Although significant advance has been made towards understanding how RNAP functions as an enzyme, including the work recognised by the Nobel Prize in Chemistry in 2006, how RNAP itself is controlled by factors that signal special cellular states and events, is still poorly understood.

We use the bacterial RNAP and its major variant sigma factor, sigma54, as a simplified model system to study how RNAP stays in an inhibited state and how activator proteins, acting remotely from where transcription will start, utilise cellular energy in the form of a high energy molecule called ATP, to convert the RNAP from an inactive enzyme to a transcriptionally competent enzyme. We have just determined the crystal structure of RNAP-sigma54 so that we now have a detailed view of what RNAP-sigma54 looks like. Our structure explains how sigma54 maintains RNAP in an inhibited state. Furthermore, we discovered many of the inhibitory strategies we see are shared to some extent by other bacterial and eukaryotic factors and reveal there are conserved hotspots in RNAP that are targeted to varying degrees by different elements and transcriptional factors to fine-tune transcription inhibition.

In this current proposed research, we plan to utilise our newly acquired knowledge, experience and reagents already generated to address fundamental questions on how this inhibited state is relieved by activator. This is likely to shed light into how RNAP in humans, plants and animals is activated. Furthermore, we want to exploit the structural features of the inhibited state to design novel antibiotics that inhibit gene transcription by attacking important sites and surfaces on the bacterial RNAP enzyme that have not been targeted before for drug therapies. Inhibiting bacterial RNAP, and hence gene transcription, is a validate antibiotic strategy e.g. in controlling TB infections, so our work should provide novel avenues for effective antibiotic development at a time when it is crucial to have new reagents to control dangerous pathogenic bacteria of humans and animals.

RNA polymerase is a fundamental cellular machinery responsible for gene transcription. RNAP is conserved from bacteria to humans. Gene transcription is a highly regulated event in response to cues in development, growth and many varying environmental stimuli. Although significant advance has been made towards understanding how RNAP functions as an enzyme, how RNAP is controlled by in cis and in trans acting factors , is still poorly understood. This is critical to RNAP sensing the outputs of signal transduction pathways.

We use the bacterial RNAP and its major variant sigma factor sigma54 as a simplified model system, important in many bacteria, to study how RNAP stays in an inhibited state and how activator proteins acting remotely from transcriptional start site utilise a AAA+ ATPase to convert it from an inactive enzyme to a transcriptionally competent enzyme. We have just determined the crystal structure of RNAP-sigma54 at 3.8 Å. Our structure explains how sigma54 maintains RNAP in an inhibited state. Furthermore, we discovered many of the inhibitory strategies are shared to some extent by other bacterial and eukaryotic factors and reveal there are conserved hotspots in RNAP that are targeted to varying degrees by different elements and transcriptional factors to fine-tune transcription inhibition. Here, we plan to utilise our newly acquired knowledge, expertise and reagents already generated to address fundamental mechanistic questions of how this inhibited state is relieved by AAA+ activators. Outcomes will also shed light on AAA+ proteins and how other RNAPs are activated. Furthermore, we want to exploit the structural features of the inhibited state to design novel antibiotics that inhibit gene transcription. Bacterial RNAP is a validated antimicrobial target,and some of the controlling hotspots we identified are not targeted by current antibiotics. So our work should provide novel avenues for new effective antibiotic developments against pathog

Key groups who will be impacted upon by the proposed research are:

(i) Academics: The academic sector will be the main short to medium term beneficiary, as the proposed research will provide knowledge, reagents and new structures of a pervasive ATPase driven gene regulation response system in E. coli, a major studied bacterium widely used to unravel the basic life processes for many decades. Furthermore, the project will provide a clear opportunity for career development and training of individuals, both nationally and internationally.Importantly in vitro mechanistic data from new structures and biochemistry will be used to help tackle controlling gene expression in vivo for antimicrobials developments .
(ii) Society at large: Benefits to society at large will be twofold: In the short term, the proposed project will provide employment and training for individuals at the postdoctoral level providing experience of project design, management as well as its high level scientific implementation, thereby directly contributing to the national economy. The interdisciplinary nature of the proposed research will greatly enhance training of the associated PDRAs, especially with respect to their ability to work within large interdisciplinary teams and deploy cutting edge approaches. Longer term benefits include impacts on health care through stimulating the formulation of new antimicrobials and refining the usage of existing ones.
iii) Industry: The industrial sector is another potential long-term beneficiary. The proposed research will generate knowledge that could potentially be exploited for new product development by the biotech and agri tech industries (e.g. against therapeutically proven antimicrobial targets). Research results could potentially identify novel targets for therapeutic intervention at protein/RNA, protein/protein and protein/DNA interaction level. The IC Business Development teams would be a valuable resource in supporting any (long term) future commercial development arising from this research. Similarly, this would benefit from the expertise offered by IC Innovations teams in the area of translating research into marketable products.
iv) Government: One of the remits of the new IC Institute for Global Health is to translate new scientific knowledge into applications to improve global health by influencing international policy. Expertise offered by the IC Institute for Global Health could therefore be exploited for using discoveries made as a result of the proposed research to inform future health care policies.

Exploitation and Application: A number of structures exist within ICL for exploitation of knowledge gained and the development of beneficial applications. For example, we could make use of the expertise offered by ICL Innovations teams in the area of translating research into marketable products. In addition, we have the opportunity to benefit from input and advice from IC Drug Discovery Centre's multi-disciplinary team whose remit is to translate early research into drug discovery projects. Results from the project will provide opportunities for novel drug-target discovery centered around protein/RNA, protein/protein and protein/DNA interactions.

For drug discovery, and noting the diminishing content of the pipelines that feed this aim, one possibility is that, as a result of the proliferation of technologies intended to enable drug discovery, the basic biological questions are being overlooked or ignored. Technological development in high throughput target identification, screening, library synthesis, and validation have their place, but they are essentially just tools, and a clear understanding of the underlying biology is paramount.This project affords such a deep mechanistic understanding of cellular responses that can then frame new approaches to drug discovery.