Final Report Summary - MTP (Mechanisms of Transcription Proofreading)
Transcription, the copying information coded in DNA into RNA, is the first step in the realization of genetic information. In all living organisms transcription is performed by multi-subunit RNA polymerases, enzymes that are highly conserved in evolution from bacteria to humans. The accuracy and processivity of RNA synthesis by RNA polymerase is essential for errorless and smooth flow of genetic information and, as a result, for the proper functioning of the cell. Transcription malfunctions, such as infidelity, are linked with various human diseases, such as cancers and Alzheimer’s. Transcription is also an attractive target for antimicrobials. The mechanisms that ensure accuracy of transcription remained largely unknown. The ERC funded project allowed us to make important inroads into transcription fidelity and processivity and some other important molecular aspects of gene expression.
To investigate the molecular mechanisms involved in RNA synthesis and their conservation in evolution, we designed in vitro transcription systems for RNA polymerases from various organisms including major human pathogens (RNA polymerases I, II and III from eukaryotic cells Saccharomyces cerevisiae, archaeal RNA polymerase, and RNA polymerases from bacteria Thermus aquaticus, Streptococcus pneumonia, Bacillus subtilis, Escherichia coli, Synechocystis sp.).
By using these systems, we uncovered the functions of a newly discovered catalytic domain of the active centres of RNA polymerase, called the Trigger Loop. We found that the Trigger Loop works as a major determinant of catalysis and fidelity of RNA synthesis. It participates in a several-check-point mechanism that discriminates against non-cognate substrates. Also Trigger Loop catalyses the removal of erroneously incorporated substrates.
Earlier we showed that the transcript itself can assist bacterial RNA polymerase to excise nucleotides incorrectly incorporated into RNA. Now we showed that the same mechanism is used by eukaryotic RNA polymerase II, suggesting that this is an ancient mechanism that emerged before divergence of bacteria and eukaryotes.
We further described how the transcript, the Trigger Loop mentioned above and a specialized cleavage factor cooperate to excise the erroneously incorporated nucleotides. This also revealed a unique ability of the RNA polymerase active centre to change synthesizing and proofreading activities in response to misincorporation events. We showed that such switch is critical to prevent RNA polymerase traffic jams in major human pathogen S. pneumoniae.
Our discoveries were not limited to fidelity of transcription and stretch into the directly and indirectly associated topics. We discovered a novel mechanism of recognition of sequences of nucleic acids by RNA polymerases, which, for example, is involved in regulation of transcription of virulence factors production in some bacteria and regulation of transcription and replication of HIV-1. We uncovered a mysterious mechanism of termination of transcription by eukaryotic RNA polymerase III, which unifies termination mechanisms between bacteria and eukaryotes. We described the mode of action of antibiotic tagetitoxin, which also made important insights into the mechanism of translocation of RNA polymerase along the DNA. We also discovered the unexpected mechanisms of action of two bacterial toxins which belong to the cellular systems that determine persistence to antibiotics.
The mentioned and some other unique experimental systems designed in our study are now applied by our collaborators from all around Europe for cutting-edge techniques which promise to further open up the field. Furthermore, we use our experimental systems to study new antibiotics targeting transcription in collaboration with local bio-tech company.
To investigate the molecular mechanisms involved in RNA synthesis and their conservation in evolution, we designed in vitro transcription systems for RNA polymerases from various organisms including major human pathogens (RNA polymerases I, II and III from eukaryotic cells Saccharomyces cerevisiae, archaeal RNA polymerase, and RNA polymerases from bacteria Thermus aquaticus, Streptococcus pneumonia, Bacillus subtilis, Escherichia coli, Synechocystis sp.).
By using these systems, we uncovered the functions of a newly discovered catalytic domain of the active centres of RNA polymerase, called the Trigger Loop. We found that the Trigger Loop works as a major determinant of catalysis and fidelity of RNA synthesis. It participates in a several-check-point mechanism that discriminates against non-cognate substrates. Also Trigger Loop catalyses the removal of erroneously incorporated substrates.
Earlier we showed that the transcript itself can assist bacterial RNA polymerase to excise nucleotides incorrectly incorporated into RNA. Now we showed that the same mechanism is used by eukaryotic RNA polymerase II, suggesting that this is an ancient mechanism that emerged before divergence of bacteria and eukaryotes.
We further described how the transcript, the Trigger Loop mentioned above and a specialized cleavage factor cooperate to excise the erroneously incorporated nucleotides. This also revealed a unique ability of the RNA polymerase active centre to change synthesizing and proofreading activities in response to misincorporation events. We showed that such switch is critical to prevent RNA polymerase traffic jams in major human pathogen S. pneumoniae.
Our discoveries were not limited to fidelity of transcription and stretch into the directly and indirectly associated topics. We discovered a novel mechanism of recognition of sequences of nucleic acids by RNA polymerases, which, for example, is involved in regulation of transcription of virulence factors production in some bacteria and regulation of transcription and replication of HIV-1. We uncovered a mysterious mechanism of termination of transcription by eukaryotic RNA polymerase III, which unifies termination mechanisms between bacteria and eukaryotes. We described the mode of action of antibiotic tagetitoxin, which also made important insights into the mechanism of translocation of RNA polymerase along the DNA. We also discovered the unexpected mechanisms of action of two bacterial toxins which belong to the cellular systems that determine persistence to antibiotics.
The mentioned and some other unique experimental systems designed in our study are now applied by our collaborators from all around Europe for cutting-edge techniques which promise to further open up the field. Furthermore, we use our experimental systems to study new antibiotics targeting transcription in collaboration with local bio-tech company.