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Real-time transcription tracking

06/03/2011
Lisa Grauer

A new technique allows scientists to observe real-time DNA transcription events in yeast.

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Researchers at the Albert Einstein College of Medicine have introduced a technique that allows real-time tracking of gene transcription in yeast. Their method could provide insight into how transcription factors function endogenously when moderating transcription events in vivo.

To view transcription at the single gene level, Daniel Larson, Albert Einstein scholar and head of the systems biology of gene expression section of the National Cancer Institute, inserted DNA sequences that create RNA stem-loop structures during transcription into the promoter region of a gene. These stem loops are quickly recognized and bound by GFP-conjugated PP7 bacteriophage coat proteins that act as tracking devices on the growing mRNA transcript.

A) POL1pro-GLT1 reporter gene construct. B) Time-dependent activity of individual reporter genes, PP7-GFP and nuclear pore marker Nup49-tomato red, with arrows indicating reporter transcripts. C) Diagram of RNA polymerase progression on the gene with corresponding fluorescence intensity trace (green line) (D). Source: Science

“Because these loops become fluorescent almost immediately, we can track the movement of the RNA polymerase and mRNA transcripts in real time and with very high precision,” said Larson.

Using this method, the group directly observed and measured the kinetic properties of single and multiple RNA polymerases during initiation, elongation, and termination of transcription of a single gene. During the initiation phase, the fluorescence signal increases steadily in intensity as the RNA polymerase proceeds through the stem loop sequence. When the gene is transcribed during the elongation phase, the fluorescence intensity remains level. A decrease in localized fluorescence marks the termination phase, when the transcript diffuses away from the active site and into the nucleoplasm.

“If we follow the fluorescence of these events with very high precision, we see fluctuations in the signal as RNA is being made and as it’s leaving. By recording and analyzing these fluctuations, we can learn something about the dynamics of transcription,” said Larson.

Published in the 22 April 2011 issue of Science, the study indicated that the recruitment of a rate-limiting transcription factor governs transcription initiation in yeast and that no transcriptional memory is retained between initiation events. The study also found that the frequency of elongation events varied throughout the cell cycle, and transcription rates were dependent on the duration for transcription factors to recognize and bind DNA promoter sites.

“The idea is emerging that the inherent dynamics of transcription—such as the binding of gene-specific activators and coactivators—also encodes information,” said Larson. “If we can attain high-resolution measurements of how these genes are actually being transcribed in real time, then we can begin to really understand how transcription factors function in the nucleus.”

Larson’s ongoing work involves applying these same methods to examine transcription events in single genes in human cells. “It turns out that many of the same molecular principles apply in humans as in yeast, but the regulation of transcription is very different,” Larson noted. “So not surprisingly, transcription in humans is much more complicated than it is in yeast.”

The paper, “Real-time observation of transcription and elongation on an endogenous yeast gene,” was published 22 April 2011 in Science.