Scientists View Genome as It Turns on and Off Inside Cells
ScienceDaily (Jan. 19, 2011) — UCSF researchers have developed a new approach to decoding the vast information embedded in an organism's genome, while shedding light on exactly how cells interpret their genetic material to create RNA messages and launch new processes in the cell.
By combining biochemical techniques with new, fast DNA-sequencing technology and advanced computer technology, the team was able to examine with unprecedented resolution how a cell converts DNA into RNA -- a molecular cousin of DNA that is used in the process of creating proteins that govern most biological functions. (Credit: Image courtesy of University of California - San Francisco)
By combining biochemical techniques with new, fast DNA-sequencing technology and advanced computer technology, the team was able to examine with unprecedented resolution how a cell converts DNA into RNA -- a molecular cousin of DNA that is used in the process of creating proteins that govern most biological functions. And they did so within the cell itself, rather than in a test tube.
As a result, they were able to bridge an important gap in the understanding of what causes genes to be turned on and off. Their findings will appear in the Jan. 20 issue of the journal Nature.
The main way the genome is "read" in a cell is through its transcription into RNA, the researchers explained. Until now, scientists have been able to detect which RNAs were produced, but have had a limited view of how much of the genome was being decoded, or "transcribed," or what controls how fast these RNAs are made. The new technique enables them to watch this process directly.
"This lets you capture the cell in the process of turning the DNA into RNA at unprecedented resolution," said Jonathan S. Weissman, PhD, a professor in the UCSF Department of Cellular and Molecular Pharmacology and senior author on the paper. "Before, we were typically studying the end product. Now, we can directly watch how these RNA messages are produced in vivo."
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Nascent transcript sequencing visualizes transcription at nucleotide resolution
L. Stirling Churchman & Jonathan S. Weissman
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Nature 469, 368–373 (20 January 2011) doi:10.1038/nature09652
Received 01 June 2010
Accepted 08 November 2010
Published online 19 January 2011
Abstract
Recent studies of transcription have revealed a level of complexity not previously appreciated even a few years ago, both in the intricate use of post-initiation control and the mass production of rapidly degraded transcripts. Dissection of these pathways requires strategies for precisely following transcripts as they are being produced. Here we present an approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3′ ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution. Application of NET-seq inSaccharomyces cerevisiae reveals that although promoters are generally capable of divergent transcription, the Rpd3S deacetylation complex enforces strong directionality to most promoters by suppressing antisense transcript initiation. Our studies also reveal pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes, with the peak location occurring in good agreement with in vitro biophysical measurements. Thus, nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo.
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