期刊
NATURE STRUCTURAL & MOLECULAR BIOLOGY
卷 14, 期 9, 页码 796-806出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nsmb1280
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资金
- NIBIB NIH HHS [EB-002060, R01 EB002060] Funding Source: Medline
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.
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