| Jul 09, 2012 |
Watching DNA unzip real time
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(Nanowerk News) Completion of the Human Genome Project in 2003 has revealed all the pieces of the basic code that together make a human being. European researchers with equally ambitious goals have visualised on a single-molecule level the initiation of protein production based on the DNA code of single genes.
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The double stranded deoxyribonucleic acid (dsDNA) that makes up the genome consists of sequences corresponding to specific genes as well as nonsense sequences. Scientists now know where all the genes start and end However, they are a long way from the equally daunting task of fully characterising how proteins are produced, how the steps can be modulated and, of course, what role the proteins play in sickness and in health.
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Cells rely on a two-step process of transcription and translation in order to ‘read’ the DNA sequence of a gene and then translate this into production of a protein. Transcription involves separation of the dsDNA in the nucleus. Its initiation requires ribonucleic acid (RNA) polymerase, sort of the ‘un-zipper’ molecule.
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EU-funded researchers working on the ambitious ‘A single-molecule view of initial transcription’ (SM-Transcription) project sought to combine state-of-the-art single-molecule spectroscopic imaging with a nano-scale spectroscopic ruler to visualise extremely small changes in DNA complexes signalling the start of transcription. The model system of choice was the bacteria Escherichia coli (E. coli).
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Although many biological processes rely on the unzipping and/or zipping of small pieces of dsDNA, very few techniques are currently available that can analyse this procedure in real time at the single-molecule level.
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The SM-Transcription team developed a modified Förster resonance energy transfer (FRET) assay to monitor a DNA fragment and indicate whether it was double stranded or single stranded. The modified (quenched) FRET (quFRET) relied on two fluorescent molecules, one on each of the two DNA strands, that exhibited quenching (a decrease) of the fluorescent signal upon contact.
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Using the quFRET assay, scientists sensed abortive initiation by bacterial RNA polymerase and elucidated the mode of action of two potent inhibitors of transcription initiation. In addition, they elucidated a number of mechanisms and conformational changes related to RNA polymerase in E. coli and provided experimental evidence for conformational fluctuations in the millisecond range using FRET rulers.
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The implications of the novel assay developed by the SM-Transcription team are mind-boggling. A complete run-through of the entire human genome and the transcription process of each protein is only the start.
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