生物谷報(bào)道：科學(xué)家們親眼目睹了活組織中的基因轉(zhuǎn)錄過(guò)程,，這一最新的研究成果在線發(fā)表在8月號(hào)的《自然—結(jié)構(gòu)和分子生物學(xué)》期刊上。
RNA聚合酶II是一種將DNA信息轉(zhuǎn)錄到信使RNA的酶,，由這種酶所實(shí)施的轉(zhuǎn)錄是基因表達(dá)的核心,，也是生物調(diào)控機(jī)制的關(guān)鍵所在,。以前,，絕大部分對(duì)這種轉(zhuǎn)錄過(guò)程的了解來(lái)自于用純化物質(zhì)在生物體外所做的試管實(shí)驗(yàn),，科學(xué)家們對(duì)RNA聚合酶在活體中的作用過(guò)程知之甚少。
利用高級(jí)熒光成像技術(shù),，Robert Singer和合作者量化測(cè)量了RNA聚合酶在活哺乳類動(dòng)物細(xì)胞中實(shí)施遺傳轉(zhuǎn)錄的動(dòng)力學(xué)過(guò)程,，從而將更高級(jí)生物中的基因轉(zhuǎn)錄機(jī)理分析提高到了一個(gè)新水平。這項(xiàng)工作的最終目標(biāo)是建立活體中基因轉(zhuǎn)錄的量化模型,。在活體組織的轉(zhuǎn)錄中,，他們發(fā)現(xiàn)了全新和意料之外的特征。
首先,，他們認(rèn)為只有大約1%的與基因相連結(jié)的RNA聚合酶參加了基因轉(zhuǎn)錄過(guò)程，并產(chǎn)生出信使RNA,。其次,，他們發(fā)現(xiàn)RNA聚合酶轉(zhuǎn)錄的速度比想象的更快，而且通常只是在周期延長(zhǎng)時(shí)才暫時(shí)停止,。
在量化認(rèn)識(shí)單個(gè)活體細(xì)胞中的轉(zhuǎn)錄機(jī)理過(guò)程中,，該新研究是一個(gè)里程碑性的貢獻(xiàn),。（科學(xué)時(shí)報(bào)）
原始出處:
Nature Structural & Molecularbiology
Published online: 5 August 2007; | doi:10.1038/nsmb1280
In vivo dynamics of RNA polymerase II transcription
Xavier Darzacq1, 2, Yaron Shav-Tal1, 3, Valeria de Turris1, Yehuda Brody3, Shailesh M Shenoy1, Robert D Phair4 & Robert H Singer1
1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
2 Laboratoire de Génétique Moléculaire, Centre National de la Recherche Scientifique, UMR-8541, Ecole Normale Supérieure, 75005 Paris, France.
3 The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel.
4 Integrative Bioinformatics, Inc., Los Altos, California 94024, USA.
Correspondence should be addressed to Robert H Singer [email protected]
We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min-1, much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.
