Final Report Summary - SM-TRANSCRIPTION (A single-molecule view of initial transcription)
During this Marie-Curie fellowship, the process of transcription, i.e. the first step from transcribing genes to functional proteins, was studied on the level of individual species. In detail, we developed novel tools to study extremely small conformational changes in protein-Deoxyribonucleic acid (DNA) complexes (see also T. Cordes et al., Biochemistry 49, 2010, 9171-9180) and were able to identify dynamic conformational changes of Ribonucleic acid (RNA) polymerase bound to DNA molecules (see also T. Cordes et al., Journal of Molecular Biology, 2011, which was submitted by the end of this reporting period).
Novel tools for the study of initial transcription were developed. Many biological processes, transcription in particular, involve opening and closing of short regions of double-stranded DNA (dsDNA). Only few techniques, however, could study transcription or replication of DNA in real time or even at the single molecule level. In the course of this Marie Curie project we developed a Förster resonance energy transfer (FRET) assay that monitored the state of DNA, observing whether it was double-stranded versus single-stranded, at a specific region within a DNA fragment. The assay utilised two closely spaced fluorophores, namely a FRET donor (Cy3B) on the first DNA strand and a FRET acceptor fluorophore (ATTO647N) on the complementary strand. Since this novel assay was based on quenching and de-quenching FRET processes, i.e. the presence or absence of contact induced fluorescence quenching, we named it a 'quenchable-FRET' assay or 'quFRET'. Using lac promoter DNA fragments, quFRET allowed us to sense transcription bubble expansion and compaction during abortive initiation by bacterial RNA polymerase. We also used quFRET to confirm the mode of action of gp2, which was a phage-encoded protein that acted as potent inhibitor of Escherichia coli (e. coli) transcription, and rifampicin, which was an antibiotic that blocked transcription initiation. Our results demonstrated that quFRET should find numerous applications in many processes involving DNA opening and closing, as well as in the development of new antibacterial therapies involving transcription.
In terms of the conformational dynamics of the open complex of e. coli RNA polymerase, it was found that transcription was initiated by the binding of RNA polymerase (RNAP) to a specific sequence on the promoter DNA. Subsequently, melting of 12 to 14 base-pairs surrounding the transcription start site formed the catalytically active RNAP-DNA open complex (RPo). During transcription initiation there was flexibility in the position at which e. coli RNAP initiated transcription. The latter was a process which gave rise to variation at the 5' end of mRNA transcripts and therefore had consequences for the regulation of gene expression. It was suggested that sampling of transcription start sites might proceed via movement of the single-stranded DNA that formed part of the transcription bubble. This DNA movement was thought to occur prior to the start of RNA synthesis and might result in 'DNA scrunching' within the transcription bubble.
In the framework of this Marie-Curie action this hypothesis was tested using single-molecule FRET (smFRET) of RPo in solution. We provided experimental evidence for conformational dynamics in RPo using different FRET rulers and labelling positions. An analysis of the FRET distributions of RPo with burst variance analysis revealed the timescale of the conformational fluctuations in RPo to be in the millisecond range. Further experiments using different subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start-sites. This investigation marked, to our knowledge, the first experimental observation of DNA dynamics in the transcription bubble of RPo and substantiated the hypothesis that these dynamics represented a DNA-scrunching based search for the transcription start site.
For further information please contact A. Kapanidis, University of Oxford (kapanidis@physics.ox.ac.uk) and T. Cordes, University of Groningen (t.m.cordes@rug.nl).
Novel tools for the study of initial transcription were developed. Many biological processes, transcription in particular, involve opening and closing of short regions of double-stranded DNA (dsDNA). Only few techniques, however, could study transcription or replication of DNA in real time or even at the single molecule level. In the course of this Marie Curie project we developed a Förster resonance energy transfer (FRET) assay that monitored the state of DNA, observing whether it was double-stranded versus single-stranded, at a specific region within a DNA fragment. The assay utilised two closely spaced fluorophores, namely a FRET donor (Cy3B) on the first DNA strand and a FRET acceptor fluorophore (ATTO647N) on the complementary strand. Since this novel assay was based on quenching and de-quenching FRET processes, i.e. the presence or absence of contact induced fluorescence quenching, we named it a 'quenchable-FRET' assay or 'quFRET'. Using lac promoter DNA fragments, quFRET allowed us to sense transcription bubble expansion and compaction during abortive initiation by bacterial RNA polymerase. We also used quFRET to confirm the mode of action of gp2, which was a phage-encoded protein that acted as potent inhibitor of Escherichia coli (e. coli) transcription, and rifampicin, which was an antibiotic that blocked transcription initiation. Our results demonstrated that quFRET should find numerous applications in many processes involving DNA opening and closing, as well as in the development of new antibacterial therapies involving transcription.
In terms of the conformational dynamics of the open complex of e. coli RNA polymerase, it was found that transcription was initiated by the binding of RNA polymerase (RNAP) to a specific sequence on the promoter DNA. Subsequently, melting of 12 to 14 base-pairs surrounding the transcription start site formed the catalytically active RNAP-DNA open complex (RPo). During transcription initiation there was flexibility in the position at which e. coli RNAP initiated transcription. The latter was a process which gave rise to variation at the 5' end of mRNA transcripts and therefore had consequences for the regulation of gene expression. It was suggested that sampling of transcription start sites might proceed via movement of the single-stranded DNA that formed part of the transcription bubble. This DNA movement was thought to occur prior to the start of RNA synthesis and might result in 'DNA scrunching' within the transcription bubble.
In the framework of this Marie-Curie action this hypothesis was tested using single-molecule FRET (smFRET) of RPo in solution. We provided experimental evidence for conformational dynamics in RPo using different FRET rulers and labelling positions. An analysis of the FRET distributions of RPo with burst variance analysis revealed the timescale of the conformational fluctuations in RPo to be in the millisecond range. Further experiments using different subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start-sites. This investigation marked, to our knowledge, the first experimental observation of DNA dynamics in the transcription bubble of RPo and substantiated the hypothesis that these dynamics represented a DNA-scrunching based search for the transcription start site.
For further information please contact A. Kapanidis, University of Oxford (kapanidis@physics.ox.ac.uk) and T. Cordes, University of Groningen (t.m.cordes@rug.nl).