Characterisation of novel functions of the active centre of RNA polymerase

Realisation of genetic information requires the copying of information coded in DNA into RNA, transcription. Afterwards this information, in the form of RNA, is either directly used by the cell or decoded into proteins during translation. In all living organisms transcription is performed by multisubunit RNA polymerases, enzymes highly conserved in evolution from bacteria to humans. Though RNA polymerase has been actively investigated for 30 years, most heavily during the last decade, the mechanisms that govern its functions are still not fully understood. Especially interesting and at the same time obscure is the functioning of the active centre of RNA polymerase, one of the most ancient protein active centres on Earth. The mechanisms ensuring accuracy, and processivity of transcription are still unclear. Furthermore, it even appears that not all of the reactions that can be catalysed by the RNA polymerase active centre are known. From an applied perspective, characterisation of the active centre of RNA polymerase is also important for drug discovery, since it is the target for potent antibiotics. Recently we have described several new features of the active centre of RNA polymerase that are important for complete understanding of its molecular function. In this proposal we will characterise the newly discovered RNA ligation reaction and search for its significance in RNA polymerase function. We will also investigate the complex mechanisms responsible for coordination and stabilisation of substrates in the active centre of RNA polymerase, and for the conformational switches in the active centre that precede catalysis in all of the reactions performed by RNA polymerase. Finally, we will search for previously unrecognised mechanisms, and new reactions of RNA polymerase. This will help to build a comprehensive picture of the mechanisms involved in transcription.

We will examine the conformational change in the RNA polymerase (RNAP) active centre that was recently suggested by us. This step is thought to include 'closing' of the active centre to provide the correct environment for the reactions, and thus represents a switch from an inactive to an active state of RNAP. Investigation of a series of mutant RNAPs in different elongation complexes will provide additional evidence for the existence of this switch and for its functional importance for different reactions catalysed by RNAP during transcription, for transcriptional fidelity and processivity. We will investigate the mechanism of this switch and the respective contribution of domains of the active centre. The mechanisms whereby reactants are positioned in the active centre of RNAP, and the roles of RNAP core and sigma subunit in this process are not yet fully understood. The hypothesis based on our recent results points to the importance of fixation of the template DNA, rather than direct positioning of the participants of the reaction, as was previously thought. In this research, using experimental systems newly designed by us, we will test this hypothesis, and provide evidence for the roles of the sigma subunit, the RNAP core and the RNA-DNA hybrid in this process. RNAP synthesises RNA by incorporating NTPs into RNA one by one. We recently discovered a new reaction whereby dinucleotides are incorporated into the transcript, which is in fact an RNA ligation, catalysed by RNAP. In the research outlined in this proposal we will characterise this new reaction. We will examine the ability of RNAP to incorporate longer RNAs. We will test these abilities in vivo, and search for the potential role of these reactions in cells. There is a high degree of confidence that the proposed research will discover new features and functions of the active centre of RNAP.