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.
Bibliographical noteFunding Information:
We acknowledge the seminal contributions of S. Janicki and D. Spector in developing the cell line. We thank D. Larson and O. Bensaude for discussions on the kinetic modeling, K. Neugebauer for suggesting the use of camptothecin, S. Buhl (Albert Einstein College of Medicine) for hybridoma supernatants containing the H14 and H5 antibodies, and E. Bertrand for sharing unpublished data. This work was online in open peer review in July 2006 on the Nature website. This work was supported by the US National Institutes of Health, National Institute of Biomedical Imaging and Bioengineering, grant EB-002060 to R.H.S. Y.S.-T. is the Jane Stern Lebell Family Fellow in Life Sciences at Bar-Ilan University.