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【bio-news】用于研究转录的新技术
New Technique Focuses on Transcription
HHMI
Converting the code contained in genes to the proteins that make up a living organism is one of the most important and highly regulated tasks performed within a cell. A new tool from Howard Hughes Medical Institute (HHMI) scientists is offering researchers a new ability to freeze that process at its earliest stage and examine the details of how it occurs, as well as how it can be sped up or slowed down.
The new technique, described in a paper published in Nature on January 20, 2011, has enabled the HHMI scientists to make fundamental discoveries about the process of transcription, in which the double-stranded DNA of our genes is transcribed into an intermediate form made of single-stranded RNA. “It provides a powerful tool for exploring how a cell turns transcription of genes on and off,” says HHMI investigator Jonathan S. Weissman, senior author of the study and a professor of cellular and molecular pharmacology at the University of California at San Francisco.
“It provides a powerful tool for exploring how a cell turns transcription of genes on and off.”
Jonathan S. Weissman
Gene transcription begins when the stretch of DNA containing a gene untwists, exposing its two nucleic-acid strands. Next, a specialized enzyme complex known as an RNA polymerase (RNAP) attaches to a promoter sequence on one of these strands. Like a mobile robot-factory, the RNAP begins to work its way from the promoter sequence along the DNA strand, knitting a mirror-image strand of RNA as it goes – the nascent transcript. At the correct end-point, the RNAP disengages from the DNA and releases the newborn RNA transcript into the nucleus, where it is sliced and spliced and otherwise chemically modified into a mature messenger RNA strand, for eventual delivery to the protein-making machinery outside the nucleus.
As busy as that sequence of events may seem, it is a greatly abbreviated version of what really happens, Weissman says. New research tools developed over the past few years have revealed, for example, that RNAP often pauses during transcription, and even backtracks. “Sometimes transcription even goes in the ‘wrong’ direction, producing mirror-image, or antisense, strands of RNA,” says L. Stirling Churchman, a postdoctoral fellow in Weissman's lab who is first author of the new paper. “These antisense strands are usually degraded quickly, but why they’re produced, and what functions they have, if any, are unknown.”
Gaps in scientists’ knowledge about transcription can be blamed, in large part, on the limited tools available for studying the process. Existing tools can isolate nascent transcripts from the rest of the RNA in a cell nucleus, but they can’t easily provide accurate location and sequence information for those transcripts in live cells.
In Churchman and Weissman’s new technique, which they call Native Elongating Transcript Sequencing, or NET-seq, they flash-freeze cells in liquid nitrogen to stop all the activity inside the cells. After applying enzymes to break up the cellular DNA into manageable lengths, they use RNAP-detecting antibodies to separate out the DNA fragments that are still attached to nascent transcripts. With modern, fast sequencing techniques, they then can find the nucleic-acid sequences of these RNA transcripts – and match them to the known cellular DNA sequence to determine the transcripts’ precise locations. By applying this technique to many cells, they also can determine all the points on a cell’s genome where transcription activity was busiest at the moment the cell was frozen.
“We almost immediately had compelling evidence that it works,” says Churchman, “because the RNA transcripts we isolated had the raw sequences that we would expect from nascent transcripts, but that are missing from mature messenger RNA.”
The precision of the NET-seq technique also enabled Churchman and Weissman to quickly make new discoveries about gene transcription. In one set of experiments, they found that normal transcription happens much more often than the “wrong-way” or antisense transcription previously reported by other researchers.
In cells, strands of DNA are wrapped around support-structure proteins known as histones. By tinkering with cells’ histone-related genes and using NET-seq to measure the effects on transcription, the researchers determined that a certain chemical mark on histones effectively makes the antisense transcription possible. “We found that under normal circumstances this mark is removed by a complex called Rpd3S, thus helping to suppress the antisense transcription,” says Weissman.
By looking at the precise locations of RNAP molecules when their test cells were frozen, Churchman and Weissman also could determine the places on the genome where RNAPs tend to pause and backtrack during transcription. Some work already had been done in this area. In 2009, for example, researchers in the labs of HHMI investigators Carlos Bustamante at the University of California at Berkeley and Michelle Wang at Cornell University reported having analyzed individual strands of DNA with single-molecule biophysical techniques. Both groups found evidence suggesting that RNAP pauses at places where the DNA it is reading comes into close contact with supporting histone proteins. “With NET-seq we could make high-resolution measurements of the RNAP locations across the entire genome of a cell, for many cells, and in this way powerfully confirmed these previous findings,” says Churchman.
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作者:admin@医学,生命科学 2011-01-24 13:15
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