Single-molecule tracking of the transcription cycle by sub-second RNA detection.
Single-molecule tracking of the transcription cycle by sub-second RNA detection.
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作者：
Zhang Z;Revyakin A;Grimm JB;Lavis LD;Tjian R
Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (∼10e7 M−1s−1), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001 The body produces proteins by transcribing DNA (genes) to make messenger RNA, which is then translated to make a protein. Transcription begins when an enzyme called RNA polymerase binds to the DNA and catalyzes the process by which genetic information from the double helix is copied to a complementary RNA transcript, which subsequently becomes the messenger RNA. Because a living cell usually contains only one or a few copies (alleles) of a given gene, molecular fluctuations play a crucial role in cellular transcription. Therefore, studying transcription kinetics at the level of single molecules may provide critical insights into how cells deal with—or even take advantage of—molecular fluctuations. A number of different single-molecule techniques can be used to follow transcription, but these techniques are often relatively slow compared to transcription in living cells, or they suffer from other problems such as only being able to study one step in the transcription process. Now, Zhang, Revyakin et al. have systematically devised a technique called ‘fastFISH’ that is fast enough to track the production of single RNA molecules directly and instantaneously. FastFISH builds on an existing technique called FISH—short for fluorescence in situ hybridization—in which fluorescent molecules are attached to single strands of DNA or RNA. These single strands pair with specific regions of complementary DNA or RNA molecules, and they can be visualized with a fluorescence microscope. However, conventional FISH is a ‘snap-shot’ technique that is not suitable for making real-time observations under physiological conditions. FastFISH relies on single strands of fluorescently labeled DNA and RNA that bind to complementary strands of DNA or RNA extremely quickly, even under physiological conditions, because they contain only three of the four ‘regular’ nucleotides that make up DNA or RNA. As a proof of principle, Zhang, Revyakin et al. used fastFISH to study the kinetics of transcription by the bacteriophage T7 RNA polymerase and were able to measure multiple stages of the transcription cycle in a single-molecule experimental setup. By allowing each stage of transcription to be tracked in real-time at the level of single-molecules, fastFISH will permit a more in-depth analysis of the factors that regulate how genes are expressed as proteins in our cells. Moreover, the ability to design single-strand probes that bind rapidly to DNA and RNA targets could have many additional applications, including new strategies for more efficient gene silencing. DOI: http://dx.doi.org/10.7554/eLife.01775.002
